Treatment of friedreich&#39;s ataxia using histone deacetylase inhibitors

ABSTRACT

The invention provides methods of treating Friedreich&#39;s ataxia and other neurodegenerative or neuromuscular conditions using histone deacetylase inhibitors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/862,727, filed Apr. 15, 2013, which is a continuation of U.S. patentapplication Ser. No. 12/773,032, filed May 4, 2010, which is acontinuation of U.S. patent application Ser. No. 11/595,779, filed Nov.10, 2006, which claims priority from U.S. Provisional Application Ser.No. 60/735,483, filed Nov. 11, 2005; U.S. Provisional Application Ser.No. 60/838,908, filed Aug. 18, 2006; and U.S. Provisional ApplicationSer. No. 60/823,051, filed Aug. 21, 2006, all of which are specificallyincorporated herein by reference in their entireties.

GOVERNMENT FUNDING

This invention was made with Government support under Grant Nos.NS048989, NS055158 and NS055781 awarded by the National Institutes ofHealth. The United States Government has certain rights in thisinvention.

TECHNICAL FIELD

The invention relates to histone deacetylase (HDAC) inhibitors and theiruses as therapeutics.

BACKGROUND

Friedreich's ataxia (FRDA) is the most prevalent inherited ataxia inCaucasians (see Pandolfo (1999) Semin. Neurol. 19:311). Individuals withFRDA have a deficiency of the mRNA encoding frataxin, a highly conserved210 amino acid nuclear-encoded, mitochondrial protein that is thought tobe involved in iron homeostasis, storage and transfer of iron-sulfurclusters to partner proteins such as aconitase (see Bulteau et al.(2004) Science 305:242; Seznec et al. (2005) Hum. Mol. Genet. 14:463;Calabrese et al. (2005) J. Neurol. Sci. 233:145).

Frataxin insufficiency leads to progressive spinocerebellarneurodegeneration resulting in gait and hand in-coordination, slurredspeech, muscle weakness and sensory loss with extraneural scoliosis,cardiomyopathy and diabetes. Generally within 15 to 20 years after thefirst appearance of symptoms, an affected individual is confined to awheelchair and in later stages, become completely incapacitated. Mostaffected individuals die in early adulthood of heart disease. Althoughantioxidant- and iron-chelator-based strategies have been used to treatFRDA, these strategies only treat the symptoms of the disease and notthe cause, i.e. frataxin deficiency. Therefore, there is a need todevelop molecules that could restore frataxin protein expression for thetreatment of a neurological condition such as FRDA.

In addition, the DNA abnormality found in 98% of FRDA patients is theunstable hyper-expansion of a GAA triplet repeat in the first intron ofthe frataxin gene (see Campuzano et al., Science 271:1423 (1996)).Triplet repeat expansion in genomic DNA is associated with many otherneurodegenerative and neuromuscular diseases including, withoutlimitation, myotonic dystrophy, spinal muscular atrophy, fragile Xsyndrome, Huntington's disease, spinocerebellar ataxias, amyotrophiclateral sclerosis, Kennedy's disease, spinal and bulbar muscular atrophyand Alzheimer's disease. Triplet repeat expansion may cause disease byaltering gene expression. For example, in Huntington's disease, thespinocerebellar ataxias, fragile X syndrome and myotonic dystrophy,expanded repeats lead to gene silencing. Therefore, there is a need todevelop molecules that could restore the normal function of genes inneurological diseases.

SUMMARY OF THE INVENTION

The invention provides small molecules that could be used to treat aneurological disease such as FRDA. The invention provides small moleculeinhibitors that are effective in restoring the normal function of agene, e.g. restoring transcription of frataxin mRNA. The presentinvention involves the discovery that lymphocytes from FRDA patientsthat have been incubated with histone deacetylase (HDAC) inhibitors showelevated levels of acetylated histones. In addition, the inventionconcerns the discovery that the HDAC inhibitor BML-210 and other novelHDAC inhibitors have the effect of increasing frataxin mRNA inlymphocytes from FRDA patients. Accordingly, the invention is directedto pharmaceutical compositions of HDAC inhibitors and their use astherapeutics for chronic and acute neurological diseases such as, forexample, Friedreich's ataxia. The invention is also directed to novelHDAC inhibitors, as well as novel methods for their synthesis.

Accordingly, in one embodiment, the invention provides a compound offormula Ia:

wherein:

-   -   n is 2 to about 10;    -   R¹ is aryl or heteroaryl;    -   R² is aryl or heteroaryl;    -   R^(a) and R^(b) are each independently H, alkyl, aryl,        heteroaryl, or a nitrogen protecting group;

wherein any alkyl, aryl or heteroaryl is optionally substituted with 1to 3 substituents selected from the group consisting of hydroxy, amino,nitro, cyano, halo, alkyl, trifluoromethyl, alkoxy, aryl, carboxyl,carboxy ester, carboxamide, and NR^(c)R^(d);

wherein R^(c) and R^(d) are each independently hydrogen, alkyl, orC(═O)OR^(e) wherein R^(e) is H or alkyl, and wherein the ester group ofthe carboxy ester is an alkyl group;

or a salt thereof;

provided that when R¹ is phenyl and n is 3-6, R² is not 2-aminophenyl;and

provided that when R¹ is 2-aminophenyl and n is 3-6, R² is not phenyl.

The compounds of formula Ia are HDAC inhibitors.

In another embodiment, the invention provides methods for preparingcompounds of formula I:

wherein:

-   -   n is 2 to about 10;    -   R¹ is aryl or heteroaryl;    -   R² is aryl or heteroaryl;    -   R^(a) and R^(b) are each independently H, alkyl, aryl,        heteroaryl, or a nitrogen protecting group;

wherein any alkyl, aryl or heteroaryl is optionally substituted with 1to 3 substituents selected from the group consisting of hydroxy, amino,nitro, cyano, halo, alkyl, trifluoromethyl, alkoxy, aryl, carboxyl,carboxy ester, carboxamide, and NR^(c)R^(d);

wherein R^(c) and R^(d) are each independently hydrogen, alkyl, orC(═O)OR^(e) wherein R^(e) is H or alkyl, and wherein the ester group ofthe carboxy ester is an alkyl group;

or a salt thereof.

According to the methods of the invention, compounds of formula I may beprepared by contacting a compound of formula V:

with one or more coupling agents and a compound of formula VI:R²—NH(R^(b))  (VI)to provide the compound of formula I. The compound of formula V may beprepared by contacting a compound of formula III:

with a compound of formula IV:R¹—NH(R^(a))  (IV)to provide the compound of formula V. The compound of formula III may beprepared by contacting a compound of formula II:

with a dehydrating agent to provide the compound of formula III.

In another embodiment, the invention provides pharmaceuticalcompositions that include a compound of formula I in combination with apharmaceutically acceptable carrier. The pharmaceutical composition maybe suitable for oral administration. Pharmaceutical compositionssuitable for oral administration can be in the form of a tablet,capsule, or elixir. The pharmaceutical compositions can also be suitablefor parenteral administration such as by intravenous, intraperitoneal orsubcutaneous administration. The pharmaceutical composition can also bein the form of a sustained-release formulation.

The pharmaceutical compositions can include an amount of the compound offormula I that is effective to increase frataxin mRNA levels in a cell.The cell can be a mammalian cell. The mammalian cell can be a human cellsuch as a lymphocyte, cardiomyocyte or neuronal cell.

The invention also provides an article of manufacture that includes thecompound of formula I contained within packaging materials that have alabel indicating that the compound of formula I can be used for treatingFriedreich's ataxia.

In another embodiment, the invention provides a method of treating, orpreventing or delaying the onset of, a neurodegenerative orneuromuscular condition in a mammal such as a human. The method involvesadministering to the mammal a compound of formula I in an amounteffective to alter the level of histone acetylation in the mammal. Thecompound of formula I may be administered orally or parenterally. Themethod may also include identifying the mammal as one suffering from, orat risk for, the neurodegenerative or neuromuscular condition. Theneurodegenerative condition may be Huntington's disease, spinocerebellarataxia, Friedreich's ataxia, Fragile X syndrome, Kennedy's disease,spinal and bulbar muscular atrophy, amyotrophic lateral sclerosis andAlzheimer's disease. The neuromuscular condition may be spinal muscularatrophy or myotonic dystrophy. Thus, in one aspect, the inventionprovides a method of treating, or preventing or delaying the onset of,Friedreich's ataxia in a mammal. This method involves administering tothe mammal a compound of formula I in an amount effective to increasefrataxin mRNA in the mammal. The method may include identifying themammal as one suffering from or at risk for Friedreich's ataxia. Amammal suffering from or at risk for Friedreich's ataxia may beidentified by determining the length, extent or number of expansion of aGAA triplet repeat in intron 1 of the frataxin gene. The mammal may alsobe identified by determining the level of frataxin mRNA or protein.

Definitions

The following definitions are used, unless otherwise described. Specificand preferred values listed below for radicals, substituents, andranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents. Alkyl, alkoxy, alkenyl, and the like denote both straightand branched groups.

As referred to herein, the group “alkyl” refers to a linear or branchedhydrocarbon radical that is optionally unsaturated and optionallysubstituted with functional groups as described herein. The alkyl groupcan contain from 1 to about 20 carbon atoms. For example and withoutlimiting the scope of the invention, alkyl can be methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, 3-pentyl,hexyl, heptyl, or octyl. In one embodiment, alkyl is preferably(C₁-C₈)alkyl. In another embodiment, alkyl is preferably (C₁-C₄)alkyl.

In embodiments where an alkyl group is unsaturated, the alkyl group isan alkenyl group. Alkenyl groups can be, for example, vinyl, 1-propenyl,2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl,5-hexenyl, 7-octenyl, and branched isomers thereof.

As referred to herein, the group “alkoxy” refers to an optionallysubstituted alkyl group that is substituted with an oxygen radical.Alkoxy can be, for example, methoxy, ethoxy, propoxy, isopropoxy,butoxy, iso-butoxy, sec-butoxy, pentoxy, 2-pentoxy, 3-pentoxy, orhexyloxy.

As referred to herein, “aryl” refers to a monovalent aromatichydrocarbon radical of 6-18 carbon atoms derived by the removal of onehydrogen atom from a single carbon atom of a parent aromatic ringsystem. Aryl groups are typically made up of 6-10 carbon atoms andadditionally can possess optional substituents as described herein.Typical aryl groups include, but are not limited to, radicals derivedfrom benzene, naphthalene, anthracene, biphenyl, and the like.

As referred to herein, “heteroaryl” refers to a monocyclic, bicyclic, ortricyclic ring system containing one, two, or three aromatic rings andcontaining at least one (typically one to about three) nitrogen, oxygen,or sulfur atoms in an aromatic ring. Heteroaryl groups can possessoptional substituents as described herein.

Examples of heteroaryl groups include, but are not limited to,2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl,benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl, cinnolinyl,dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl,indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl,perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl,pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl,and xanthenyl.

In one embodiment the term “heteroaryl” denotes a monocyclic aromaticring containing five or six ring atoms containing carbon and 1, 2, 3, or4 heteroatoms independently selected from non-peroxide oxygen, sulfur,and N(Z) wherein Z is absent or is H, O, alkyl, phenyl or benzyl. Inanother embodiment heteroaryl denotes an ortho-fused bicyclicheterocycle of about eight to ten ring atoms derived therefrom,particularly a benz-derivative or one derived by fusing a propylene,trimethylene, or tetramethylene diradical thereto.

As referred to herein, “optionally” substituted group refers to thesubstitution of a group in which one or more hydrogen atoms are eachindependently replaced with a non-hydrogen substituent. Groups that areoptionally substituted are typically substituted with one to fivesubstituents. In other embodiments, optionally substituted groups aresubstituted with one to three substituents. Typical substituentsinclude, but are not limited to, —X, —R, —O⁻, ═O—OR, +S⁻, —SR, —S(═O)R,—S(═O)₂R, —S(═O)₂O⁻, —S(═O)₂OH, —OS(═O)₂OR, —S(═O)₂NR, —NR₂, —N⁺R₃, ═NR,—N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, NC(═O)R, —CX₃, —C(O)O⁻, —C(═O)R,—C(O)OR, —C(═O)X, —C(═O)NRR, —C(S)R, —C(S)OR, —C(O)SR, —C(S)SR,—C(S)NRR, —C(NR)NRR, —CN, —OCN, —SCN, —OP(═O)(OR)₂, —P(═O)(OR)₂,—P(═O)(O⁻)₂, —P(═O)(OH)₂, where each X is independently a halogen (F,Cl, Br, or I); and each R is independently H, alkyl, aryl, aheterocycle, or a protecting group. When the substituent is attached toa group by two bonds (e.g., by a “double bond”), two hydrogen atoms arereplaced by the substituent.

As to any of the above groups that contain one or more substituents, itis understood, of course, that such groups do not contain anysubstitution or substitution patterns which are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds.

As used herein, the term “nitrogen protecting group” refers to any groupwhich, when bound to a nitrogen group, can serve to prevent undesiredreactions from occurring at this group and which can be removed byconventional chemical or enzymatic steps to reestablish the freenitrogen (e.g, a —NH— group or a —N═ group) at a later stage.

The hydroxyl, carboxyl, amino, and amido groups of the compoundsdescribed herein can include optional protecting groups. Suitableprotecting groups are known to those skilled in the art. A large numberof protecting groups and corresponding chemical cleavage reactions thatcan be used in conjunction with the compounds of the invention aredescribed in Protective Groups in Organic Synthesis, Theodora W. Greene(John Wiley & Sons, Inc., New York, 1991, ISBN 0-471-62301-6), which isincorporated herein by reference in its entirety. Included therein arehydroxyl protecting groups, carboxylic acid protecting groups, andamide-forming groups. In particular, see Chapter 1, Protecting Groups:An Overview, pages 1-20; Chapter 2, Hydroxyl Protecting Groups, pages21-94; Chapter 4, Carboxyl Protecting Groups, pages 118-154; and Chapter5, Carbonyl Protecting Groups, pages 155-184. See also Kocienski, PhilipJ.; Protecting Groups (Georg Thieme Verlag Stuttgart, N.Y., 1994), whichis incorporated herein by reference in its entirety. Some specificprotecting groups that can be employed in preparing the compounds of theinvention are discussed below in the section describing the use ofprotecting groups.

As used herein, a “base” refers to any molecule, ion, or other entitythat acts as a proton acceptor. A base can be an organic compound or ionwith an unshared electron pair. Typical bases include mono-, di-, andtri-alkyl substituted amines. A base can also be an inorganic compoundor ion, such as a metal oxide or metal hydroxide. Bases used in organicsynthesis are well known to those of skill in the art. Many bases aredisclosed in, for example, the Aldrich Handbook of Fine Chemicals,2003-2004 (Milwaukee, Wis.).

As used herein, “solvent” refers to a substance, usually a liquid,capable of dissolving another substance, e.g., a solid substance,semi-solid substance, or a liquid. Typical solvents include water andorganic solvents. It is appreciated by those of skill in the art thatthe solvent should not chemically react with any of the startingmaterials or reagents present in the reaction mixture, to anysignificant degree, under the reaction conditions employed.

As used herein, “solvent system” refers to a medium that includes one ormore solvents. A solvent system can be homogeneous (miscible solvents)or heterogeneous (e.g, an organic/aqueous system).

As used herein, “reflux” refers to the process of boiling a liquidsolvent system in a vessel, for example, a vessel attached to acondenser, so that the vapors of the solvent system continuouslycondense for reboiling.

As used herein, “purifying” refers to the process of ridding a substrate(e.g., crystals, an amorphous solid, a liquid, or an oil) of impurities.Suitable methods of purifying include, for example, filtering, washing,recrystallizing and drying, distilling, and chromatography.

As used herein, the terms “isolated” and “purified” refer to substancesthat are substantially free of other agents, for example, at least about90%, at least about 95%, at least about 98%, or, at least about 99% pureby weight.

As used herein, “anhydrous” refers to a substance that contains lessthan about 10 wt. % water, less than about 1 wt. % water, less thanabout 0.5 wt. % water, less than about 0.1 wt. % water, or less thanabout 0.01 wt. % water. Anhydrous conditions refer to reactionconditions that have less than about 2 wt. % water, less than about 1wt. % water, less than about 0.5 wt. % water, less than about 0.1 wt. %water, or less than about 0.01 wt. % water present.

As used herein, “contacting” refers to the act of touching, makingcontact, or of bringing into immediate proximity. Compounds aretypically contacted by forming a solution in a suitable solvent system.

In describing the details of the compounds, compositions, and otherlimitations, the numerical ranges given herein are those amounts thatprovide functional results in the composition. Thus, ranges aregenerally introduced with the term “about” to indicate a certainflexibility in the range. For example, the term “about” can refer to +/−one integer from a given number or the upper or lower limit of range. Inother embodiments, the term “about” can refer to +/− two integers from agiven number or the upper or lower limit of range. The term “about” canalso refer to +/−20% of a given number or numerical range. In otherembodiments, the term “about” can refer to +/−10%, or +/−5% of a givennumber or numerical range. In yet other embodiments, the term “aboutrefers to +/−1%. In still other embodiments, the term “about” refers toexactly the given number or numerical range.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents and other referencesmentioned herein are incorporated by reference in their entirety. Thepresent specification provides selected definitions of certain terms,and these definitions are preferred relative to other definitions in theevent that there are discrepancies. In addition, the materials, methods,and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-1B show bar graphs illustrating the histone modifications onfrataxin gene chromatin. FIG. 1A shows results from immunoprecipitation(ChIP) experiments performed with the FRDA cell line (GM15850) and thenormal cell line (GM15851) using antibodies to the acetylated forms ofhuman histones H3 and H4 (acetylated at the lysine residues indicated).Primer pairs for the frataxin promoter (Pro), and regions immediatelyupstream (Up) and downstream (Down) of the GAA repeats in the firstintron of the frataxin gene were used. Relative recovery, as determinedby real-time PCR, is expressed in relation to GAPDH, and the recovery onthe Up GAA region for each antibody is set to a value of 100. Error barsare the s.e.m. (standard error of measurement) of 2-3 independentimmunoprecipitation, and each immunoprecipitation was quantified intriplicate. FIG. 1B shows ChIP results performed for the region upstreamof the GAA repeats using antibodies to histone H3 mono-, di- andtri-methylated at K9 for both the FRDA and normal cell lines. Recoveryis expressed as percent of GAPDH.

FIG. 2A-2B show bar graphs illustrating the effects of histonedeacetylase inhibitors on acetylation and frataxin mRNA in FRDA cells.FIG. 2A shows the effects of histone deacetylase inhibitors on thelevels of H3 and H4 acetylation in an FRDA lymphoid cell line (15850B).Cells were either untreated or treated with the indicated compounds for12 hours prior to isolation of acid soluble nuclear proteins, SDS-PAGEand western blotting with antibodies to total histone H4/H3 oracetylated H4/H3. The fold changes in normalized ratio of AcH4 or AcH3to total H4 or H3 are shown in the bar graph. FIG. 2B shows relativeFrataxin mRNA levels determined by quantitative RT-PCR. All values arenormalized to GAPDH mRNA levels, which were unaffected by the HDACinhibitors. Each of the HDAC inhibitors was tested at the IC₅₀ valuereported by the commercial supplier, as indicated. Error bars are s.e.m.

FIG. 3 is an autoradiogram showing that HDAC inhibitors increasefrataxin protein in the FRDA lymphoid cell line. Cells were incubatedwith the indicated concentrations of HDAC inhibitors for 4 days prior towestern blot analysis with antibody to human frataxin or actin.Equivalent amounts of total cell extract protein were loaded in eachlane. The fold changes in frataxin protein compared to untreated controlcells (denoted “Ctrl” in the figure), normalized to the actin signals,are 1.6 (2.5 μM 4c/BML-210), 3.4 (5 μM 4c), and 3.5 (2.5 μM 4b).

FIG. 4A-4C show bar graphs showing that HDAC inhibitors increasefrataxin mRNA in primary lymphocytes from FRDA patients. Frataxin mRNAlevels were determined by qRT-PCR, relative to that of GAPDH, inlymphocytes from an unaffected individual A (normal range of repeats)and his/her FRDA sibling (affected S, with frataxin alleles containingand 906 and 88 GAA repeats) (FIG. 4A); in lymphocytes from carrier C andaffected AC (801 and 597 repeats) (FIG. 4B); and in lymphocytes fromcarrier D, and affected J (550 and 530 repeats) and M (1030 and 650repeats) (FIG. 4C). The indicated concentrations of HDAC inhibitors wereincluded in the cell culture medium, and frataxin and GAPDH mRNA levelswere determined at 48 hours. Data are normalized to the frataxin mRNAlevel found in lymphocytes from the unaffected individuals (normal inFIG. 4A or carriers in FIGS. 4B and 4C, =100%). The means and standarddeviations for three independent determinations are shown.

FIG. 5A-5B show bar graphs illustrating the effects of HDAC inhibitorson histone acetylation at the frataxin gene. FIG. 5A shows that HDACinhibitor 4b increases histone acetylation at particular H3 and H4lysines on the frataxin gene. FRDA cells were treated with 4b (5 μM for96 h) prior to ChIP with the indicated antibodies, and PCR was performedwith primers for the region upstream of the GAA repeats. Data are shownfor both untreated cell lines and the FRDA cells treated with 4b.Recovery is expressed as percent of GAPDH, and all values are normalizedto those for GM15851 cells. FIG. 5B shows that SAHA and TSA do notaffect histone acetylation on the frataxin gene. FRDA cells wereincubated for 96 hours with 2.5 μM SAHA or 0.1 μM TSA and processed forChIP as in FIG. 5A. Recovery is expressed relative to untreated GM15850cells, normalized for GAPDH. Error bars are s.e.m.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides small molecules that could be used to treat aneurological condition such as FRDA. The invention concerns thediscovery that lymphocytes from FRDA patients that have been incubatedwith histone deacetylase (HDAC) inhibitors show elevated levels ofacetylated histones. In addition, the invention concerns the discoverythat the HDAC inhibitors, suberoylanilide orthoaminoanilide (SAOA,BML-210) and pimeloylanilide orthoaminoanilide (PAOA), as well as novelderivatives of SAOA and PAOA, have the effect of increasing frataxinmRNA and protein expression in lymphocytes from FRDA patients.Accordingly, the invention provides pharmaceutical compositions of HDACinhibitors and their use as therapeutics for chronic and acuteneurological diseases such as, for example, Friedreich's ataxia. Theinvention also provides novel HDAC inhibitors, as well as novel methodsfor their synthesis.

Histone Deacetylase Inhibitors

The DNA abnormality found in 98% of FRDA patients is the unstablehyper-expansion of a GAA triplet repeat in the first intron of thefrataxin gene that results in a defect in transcription of the frataxingene (see Campuzano et al. (1996) Science 271: 1423-7). FRDA patientshave a marked deficiency of frataxin mRNA, and longer GAA tripletrepeats also cause a more profound frataxin deficiency. FRDA is typicalof triplet repeat diseases: normal alleles have 6-34 repeats while FRDApatient alleles have 66-1700 repeats. Longer GAA triplet repeats areassociated with earlier onset and increased severity of the disease.

The invention provides for histone deacetylase (HDAC) inhibitors thatcan restore gene function in a neurological disease that is associatedwith expansion of a triplet repeat such as FRDA. For example, a HDAC ofthe invention can increase frataxin mRNA and protein in lymphocytes fromFRDA patients. A “histone deacetylase inhibitor” is a small moleculethat binds to one or more histone deacetylase (HDAC) to modulate thelevels of acetylation of histones, non-histone chromosomal proteins, andother cellular proteins. An HDAC inhibitor of the invention may interactwith a HDAC to modulate the level of acetylation of cellular targets.

A histone deacetylase (HDAC) may be any polypeptide having featurescharacteristics of polypeptides that catalyze the removal of the acetylgroup (deacetylation) from acetylated target proteins. Featurescharacteristics of HDAC are known in the art, see, for example, Finninet al. (1999) Nature 401: 188. Thus, a HDAC may be a polypeptide thatrepresses gene transcription by deacetylating the ϵ-amino groups ofconserved lysine residues located at the N-termini of histones, e.g. H3,H4, H2A and H2B, that form the nucleosome. HDACs may also deacetylateother proteins such as p53, E2F, α-tubulin and Myo D. See Annemieke etal. (2003) Biochem. J. 370: 737. HDAC may also be localized to thenucleus or one that may be found in both the nucleus and cytoplasm.

An HDAC inhibitor of the invention may interact with any HDAC. Forexample, an HDAC inhibitor of the invention may interact with HDAC fromone of the three known classes of HDAC. An HDAC inhibitor of theinvention may interact with an HDAC of the class I or class II family ofHDAC. Class I HDACs are those that most closely resemble the yeasttranscriptional regulator RPD3. Examples of class I HDACs include HDACs1, 2, 3 and 8, as well as any HDAC that has a deacetylase domainexhibiting from 45% to 93% identity in amino acid sequence to HDACs 1,2, 3 and 8. Class II HDACs are those that most closely resemble theyeast HDA1 enzyme, and examples of class II HDACs include HDACs 4, 5, 6,7, 9 and 10. An HDAC inhibitor of the invention may also interact withthe NAD⁺-dependent family of HDACs, which most closely resemble theyeast SIR2 protein. An HDAC inhibitor of the invention may also interactwith HDACs that do not fall into one of the above classes, see e.g. Gaoet al. (2002) J. Biol. Chem. 277: 25748.

Small molecular weight HDAC inhibitors of the invention include SAOA andPAOA, derivatives of SAOA and PAOA described herein and salts thereof.Thus, HDAC inhibitors of the invention include compounds of formula I:

wherein:

-   -   n is 2 to about 10;    -   R¹ is aryl or heteroaryl;    -   R² is aryl or heteroaryl;    -   R^(a) and R^(b) are each independently H, alkyl, aryl,        heteroaryl, or a nitrogen protecting group;

wherein any alkyl, aryl or heteroaryl is optionally substituted with 1to 3 substituents selected from the group consisting of hydroxy, amino,nitro, cyano, halo, alkyl, trifluoromethyl, alkoxy, aryl, carboxyl,carboxy ester, carboxamide, and NR^(c)R^(d);

wherein R^(c) and R^(d) are each independently hydrogen, alkyl, orC(═O)OR^(e) wherein R^(e) is H or alkyl, and wherein the ester group ofthe carboxy ester is an alkyl group;

or a salt thereof.

In formula I, the alkyl, aryl or heteroaryl substitutent may be otherthan carboxyl, carboxy ester, or carboxamide.

In one embodiment, R¹ can be aryl. In another embodiment, R¹ can beheteroaryl. In other embodiments, R¹ can be phenyl, 2-aminophenyl,3-aminophenyl, 4-aminophenyl, 2-methoxyphenyl, 3-methoxyphenyl, or4-methoxyphenyl. In other embodiments R¹ can be 2-methylphenyl,3-methylphenyl, or 4-methylphenyl. In other embodiments, R¹ can be2,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl,2,4-diaminophenyl, 3,5-diaminophenyl, or 3,4,5-triaminophenyl. In stillother embodiments, R¹ can be 2-pyridinyl, 3-quinolinyl, or 8-quinolinyl.

In one embodiment, R² can be aryl. In another embodiment, R² can beheteroaryl. In certain embodiments, R² can be phenyl, 2-aminophenyl,3-aminophenyl, 4-aminophenyl, 2-methoxyphenyl, 3-methoxyphenyl, or4-methoxyphenyl. In other embodiments R¹ can be 2-methylphenyl,3-methylphenyl, or 4-methylphenyl. In other embodiments, R² can be2,4-dimethoxyphenyl, 3,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl,2,4-diaminophenyl, 3,5-diaminophenyl, or 3,4,5-triaminophenyl. In yetanother embodiment, R² can be 2-pyridinyl, 3-quinolinyl, or8-quinolinyl.

In some embodiments, R¹ and R² can be the same. In other embodiments, R¹and R² are not the same.

In one embodiment, R^(a) is H. In another embodiment, R^(b) is H. In yetanother embodiment, R^(a) is a nitrogen protecting group. In yet anotherembodiment, R^(b) can be a nitrogen protecting group.

In one embodiment, n is about 3 to about 6. In another embodiment, n is5. In yet another embodiment, n is 6.

In one embodiment, R¹ can be substituted with one or more substituents.R¹ can be substituted with one to about five, or one to about three,substituents. In one embodiment, R¹ can be substituted with two aminogroups. In another embodiment, R¹ can be substituted with two methoxygroups.

In one embodiment, R² can be substituted with one or more substituents.R² can be substituted with one to about five, or one to about three,substituents. In one embodiment, R² can be substituted with two aminogroups. In another embodiment, R² can be substituted with two methoxygroups.

Methods of Synthesis of HDAC Inhibitors

The invention also provides novel methods for the synthesis of HDACinhibitors. For example, compounds of formula I may be prepared bycontacting a compound of formula V:

with one or more coupling agents and a compound of formula VI:R²—NH(R^(b))  (VI)to provide the compound of formula I. The coupling agents may be1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (EDC) and1-hydroxy-7-azabenzotriazole (HOAt). The coupling of compounds offormula V and VI may be carried out in the presence of one or more basiccompounds. Suitable basic compounds or “bases” include alkyl amines. Thealkyl amine may be tri-alkyl substituted amines, for example,triethylamine or diisopropylethylamine. Hindered amines such as2,6-lutidine and 2,4,6-collidine may also be used in certain embodimentsof the invention.

The coupling of compounds of formula V and VI may be carried out in thepresence of a solvent system. Typical solvent systems may be one solventor more than one solvent. The solvent system may be one or more organicsolvents. Two component solvent systems include two solvents that aremiscible with one another. The solvent system may dissolve the compoundsof formula V and VI to a degree that allows the reaction to proceed tothe formation of the compound of formula I. Suitable solvents includedimethylformamide, dimethyl sulfoxide (DMSO), N-methyl pyrrolidone(NMP), tetrahydrofurane (THF), 1,4-dioxane, dichloromethane, and anyother suitable non-protic solvent.

The compound of formula I may be isolated and purified. Purificationtechniques that may be used include precipitation, filtration,recrystallization, and other forms of chromatography including, but notlimited to GC, HPLC, reverse phase chromatography, gel plate, thin layerchromatography and the like.

The invention also provides a method of preparing the compound offormula V by contacting a compound of formula III:

with a compound of formula IV:R¹—NH(R^(a))  (IV)to provide the compound of formula V. The compounds of formula III andIV may be contacted in the presence of a solvent system. The solventsystem may include one or more organic solvents. Suitable solventsinclude ether, tetrahydrofuran, and dioxane. In one embodiment, thesolvent is tetrahydrofuran.

The invention also provides a method of preparing the compound offormula III by contacting a compound of formula II:

with a dehydrating agent to provide the compound of formula III. Thedehydrating agent may be a carboxylic anhydride. In one embodiment, thecarboxylic anhydride is acetic anhydride. Other alkyl or aryl carboxylicanhydrides may also be employed in the reaction. The formation ofcompounds of formula III are typically carried out under anhydrousconditions. Anhydrous conditions may be achieved by suitable drying ofreactants, reagents, and equipment. The compound of formula II and thedehydrating agent may be heated to facilitate the formation of thecompound of formula III. The temperature of the reaction may beincreased, for example, to about 35° C., to about 40° C., to about 50°C., to about 70° C., or to about 100° C. The temperature of the reactionmay also be determined by the temperature at which the solvent systemachieves reflux. In such cases, the reaction may be to the refluxtemperature of the solvent system employed.

The synthetic protocol for preparing a compound of formula V from acompound of formula II may be illustrated as shown below in Scheme 1:

The compound of formula III can be isolated and purified. Alternatively,the compound of formula III can be converted to the compound of formulaV directly without purification.

The synthetic protocol for preparing a compound of formula I from acompound of formula V may be illustrated as shown below in Scheme 2:

These methods are intended to illustrate the nature of suchpreparations, not to limit the scope of applicable methods. HDACinhibitors of the invention may be prepared as described herein or usingany other applicable techniques of organic synthesis known in the art.Many applicable techniques not described herein are well known in theart. However, many of the known techniques are elaborated in Compendiumof Organic Synthetic Methods (John Wiley & Sons, New York), Volumes 1-6;as well as March, J., Advanced Organic Chemistry, 3^(rd) Ed. (John Wiley& Sons, New York, 1985), and Comprehensive Organic Synthesis.Selectivity, Strategy & Efficiency in Modern Organic Chemistry, in 9Volumes, Barry M. Trost, Ed.-in-Chief (Pergamon Press, New York, 1993printing).

Generally, the reaction conditions such as temperature, reaction time,solvents, work-up procedures, and the like, will be those common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically the temperatures will beabout −100° C. to about 200° C., solvents will be aprotic or proticdepending on the conditions required, and reaction times will be 1minute to 10 days. Reaction times are adjusted to achieve desiredconversions. Work-up of reactions can include removal of solvent toprovide crude products, precipitation and filtration, and/or quenchingof any unreacted reagents followed by partition between a water/organiclayer system (extraction) and separation of the layer containing theproduct.

Protecting Groups:

The term “protecting group” may refer to any group which, when bound toa hydroxyl, nitrogen, or other heteroatom, prevents undesired reactionsfrom occurring at this group and which can be removed by conventionalchemical or enzymatic steps to reestablish the hydroxyl group. Theparticular removable protecting group employed is not critical andpreferred removable hydroxyl and nitrogen protecting groups includeconventional substituents such as, for example, allyl, benzyl, acetyl,chloroacetyl, thiobenzyl, benzylidine, phenacyl, methyl methoxy, silylethers (e.g., trimethylsilyl (TMS), t-butyl-diphenylsilyl (TBDPS), ort-butyldimethylsilyl (TBS)) and any other group that can be introducedchemically onto a hydroxyl or nitrogen functionality and laterselectively removed either by chemical or enzymatic methods in mildconditions compatible with the nature of the product.

Suitable hydroxyl protecting groups are known to those skilled in theart and disclosed in more detail in T. W. Greene, Protecting Groups InOrganic Synthesis; Wiley: New York, 1981, (“Greene”) and the referencescited therein, and Kocienski, Philip J.; Protecting Groups (Georg ThiemeVerlag Stuttgart, N.Y., 1994), both of which are incorporated herein byreference in its entirety.

Protecting groups are available, commonly known and used, and areoptionally used to prevent side reactions with the protected groupduring synthetic procedures, i.e. routes or methods to prepare thecompounds by the methods of the invention. For the most part thedecision as to which groups to protect, when to do so, and the nature ofthe chemical protecting group will be dependent upon the chemistry ofthe reaction to be protected against (e.g., acidic, basic, oxidative,reductive or other conditions) and the intended direction of thesynthesis.

The protecting groups do not need to be, and generally are not, the sameif the compound is substituted with multiple protecting groups. Ingeneral, protecting groups will be used to protect functional groupssuch as carboxyl, hydroxyl, thio, or amino groups and to thus preventside reactions or to otherwise facilitate the synthetic efficiency. Theorder of deprotection to yield free, deprotected groups is dependentupon the intended direction of the synthesis and the reaction conditionsto be encountered, and may occur in any order as determined by theartisan.

Various functional groups of the compounds of the invention may beprotected. For example, protecting groups for —OH groups (whetherhydroxyl, carboxylic acid, or other functions) include “ether- orester-forming groups”. Ether- or ester-forming groups are capable offunctioning as chemical protecting groups in the synthetic schemes setforth herein. However, some hydroxyl and thio protecting groups areneither ether- nor ester-forming groups, as will be understood by thoseskilled in the art. Some are, for example, included in the discussion ofamides, discussed below.

For further detail regarding carboxylic acid protecting groups and otherprotecting groups for acids, see Greene as set forth below. Such groupsinclude by way of example and not limitation, esters, amides,hydrazides, and the like.

Ether-, Ester-, and Amide-Forming Protecting Groups:

Ester- and Amide-forming groups include: (1) carboxylester/amide-forming groups, and (2) sulfur ester-forming groups, such assulfonate, sulfate, and sulfinate. In its ester-forming role, aprotecting group typically is bound to any acidic group such as, by wayof example and not limitation, a —CO₂H group, thereby resulting in—CO₂R^(a) where R^(a) is as defined herein. Examples of protectinggroups include:

Heterocycle or aryl radicals. These groups optionally can be polycyclicor monocyclic. Examples include phenyl, spiryl, 2- and 3-pyrrolyl, 2-and 3-thienyl, 2- and 4-imidazolyl, 2-, 4- and 5-oxazolyl, 3- and4-isoxazolyl, 2-, 4- and 5-thiazolyl, 3-, 4- and 5-isothiazolyl, 3- and4-pyrazolyl, 1-, 2-, 3- and 4-pyridinyl, and 1-, 2-, 4- and5-pyrimidinyl; and

Heterocycle or aryl substituted with halo, R^(a), alkylene-O—R^(a),alkoxy, —CN, —NO₂, —OH, carboxy, carboxyester, thiol, thioester,haloalkyl (1-6 halogen atoms), alkenyl, or alkynyl. Such groups include2-, 3- and 4-alkoxyphenyl (C₁-C₁₂ alkyl), 2-, 3- and 4-methoxyphenyl,2-, 3- and 4-ethoxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and3,5-diethoxyphenyl, 2- and 3-carboethoxy-4-hydroxyphenyl, 2- and3-ethoxy-4-hydroxyphenyl, 2- and 3-ethoxy-5-hydroxyphenyl, 2- and3-ethoxy-6-hydroxyphenyl, 2-, 3- and 4-O-acetylphenyl, 2-, 3- and4-dimethylaminophenyl, 2-, 3- and 4-methylmercaptophenyl, 2-, 3- and4-halophenyl (including 2-, 3- and 4-fluorophenyl and 2-, 3- and4-chlorophenyl), 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-dimethylphenyl,2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-biscarboxyethylphenyl, 2,3-, 2,4-,2,5-, 2,6-, 3,4- and 3,5-dimethoxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-and 3,5-dihalophenyl (including 2,4-difluorophenyl and3,5-difluorophenyl), 2-, 3- and 4-haloalkylphenyl (1 to 5 halogen atoms,C₁-C₁₂ alkyl including 4-trifluoromethylphenyl), 2-, 3- and4-cyanophenyl, 2-, 3-, and 4-nitrophenyl, 2-, 3- and 4-haloalkylbenzyl(1 to 5 halogen atoms, C₁-C₁₂ alkyl including 4-trifluoromethylbenzyland 2-, 3- and 4-trichloromethylphenyl and 2-, 3- and4-trichloromethylphenyl), 4-N-methylpiperidinyl, 3-N-methylpiperidinyl,1-ethylpiperazinyl, benzyl, alkylsalicylphenyl(C₁-C₄ alkyl, including2-, 3- and 4-ethylsalicylphenyl), 2-,3- and 4-acetylphenyl,1,8-dihydroxynaphthyl (—C₁₀H₆—OH) and aryloxy ethyl[C₆-C₉ aryl(including phenoxy ethyl)], 2,2′-dihydroxybiphenyl, 2-, 3-, and4-N,N-dialkylaminophenol, —C₆H₄CH₂—N(CH₃)₂, trimethoxybenzyl,triethoxybenzyl, 2-alkyl pyridinyl(C₁₋₄ alkyl); C₄-C₈ esters of2-carboxyphenyl; and C₁₋₄ alkylene-C₃-C₆ aryl (including benzyl,—CH₂-pyrrolyl, —CH₂-thienyl, —CH₂-imidazolyl, —CH₂-oxazolyl,—CH₂-isoxazolyl, —CH₂-thiazolyl, —CH₂-isothiazolyl, —CH₂-pyrazolyl,—CH₂-pyridinyl and —CH₂-pyrimidinyl) substituted in the aryl moiety by 3to 5 halogen atoms or 1 to 2 atoms or groups selected from halogen,C₁-C₁₂ alkoxy (including methoxy and ethoxy), cyano, nitro, —OH, C₁-C₁₂haloalkyl (1 to 6 halogen atoms; including —CH₂CCl₃), C₁-C₁₂ alkyl(including methyl and ethyl), C₂-C₁₂ alkenyl or C₂-C₁₂ alkynyl; alkoxyethyl [C₁-C₆ alkyl including —CH₂—CH₂—O—CH₃ (methoxy ethyl)]; alkylsubstituted by any of the groups set forth above for aryl, in particular—OH or by 1 to 3 halo atoms (including —CH₃, —CH(CH₃)₂, —C(CH₃)₃,—CH₂CH₃, —(CH₂)₂CH₃, —(CH₂)₃CH₃, —(CH₂)₄CH₃, —(CH₂)₅CH₃, —CH₂CH₂F,—CH₂CH₂Cl, —CH₂CF₃, and —CH₂CCl₃); 2-N-morpholino-ethyl;—N-2-propylmorpholino, 2,3-dihydro-6-hydroxyindene, sesamol, catecholmonoester, —CH₂—C(O)—N(R¹)₂, —CH₂—S(O)(R¹), —CH₂—S(O)₂(R¹),—CH₂—CH(OC(O)CH₂R¹)—CH₂(OC(O)CH₂R¹), cholesteryl, enolpyruvate(HO₂C—C(═CH₂)—), and glycerol.

Further examples of protecting groups are ester moieties that, forexample, can be bonded via an oxygen of the compound of the invention to—C(O)—O—PG′ wherein PG′ is —CH₂—C(O)—N(R¹)₂, —CH₂—S(O)(R¹),—CH₂—S(O)₂(R¹), —CH₂—O—C(O)—CH₂—C₆H₅, 3-cholesteryl, 3-pyridyl,N-ethylmorpholino, —CH₂—O—C(O)—C₆H₅, —CH₂—O—C(O)—CH₂CH₃,—CH₂—O—C(O)—C(CH₃)₃, —CH₂—CCl₃, —C₆H₅, —NH—CH₂—C(O)O—CH₂CH₃,—N(CH₃)—CH₂—C(O)O—CH₂CH₃, —NHR¹, —CH₂—O—C(O)—C₁₀H₁₅,—CH₂—O—C(O)—CH(CH₃)₂, and —CH₂—CH(OC(O)CH₂R¹)—CH₂—(OC(O)CH₂R¹). Many ofthese esters can be synthesized by reacting the compound herein having afree hydroxyl (or acid group) with the corresponding halide (chloride oracyl chloride and the like) and N,N-dicyclohexyl-N-morpholinecarboxamidine (or another base such as DBU, triethylamine, CsCO₃,N,N-dimethylaniline and the like) in DMF (or other solvent such asacetonitrile or N-methylpyrrolidone). Coupling reagents can be used tofacilitate linkage of the compound and the protecting group. Otheresters can be synthesized by the methods described by Greene, or byother methods well known to those of skill in the art.

Protecting groups also includes “double ester” formingpro-functionalities such as —CH₂OC(O)OCH₃, —CH₂SCOCH₃, —CH₂OCON(CH₃)₂,dihydro-furan-2-one-5-yl, or alkyl- or aryl-acyloxyalkyl groups (linkedto oxygen of the acidic group) (see U.S. Pat. No. 4,968,788). Anotherexample is the pivaloyloxymethyl group. Other examples of usefulprotecting groups are alkylacyloxymethyl esters and their derivatives,including: 2-(adamantine-1-carboxylate)-ethyl,—CH(CH₂CH₂OCH₃)OC(O)C(CH₃)₃, —CH₂OC(O)C₁₀H₁₅, —CH₂OC(O)C(CH₃)₃,—CH(CH₂OCH₃)OC(O)C(CH₃)₃, —CH(CH(CH₃)₂)OC(O)C(CH₃)₃, —CH₂OC(O)CH₂CH(CH₃)₂, —CH₂OC(O)C₆H₁₁, —CH₂OC(O)C₆H₅, —CH₂OC(O)C₁₀H₁₅, —CH₂OC(O)CH₂CH₃,—CH₂OC(O)CH(CH₃)₂, —CH₂OC(O)C(CH₃)₃ and —CH₂OC(O)CH₂C₆H₅. Other estersthat are suitable for use herein are described in EP 632048.

One or more acidic hydroxyls can be protected. If more than one acidichydroxyl is protected then the same or a different protecting group canbe employed, e.g., the esters may be different or the same, or a mixedamidate and ester may be used.

Typical nitrogen and hydroxy protecting groups described in Greene(pages 14-118) include substituted methyl and alkyl ethers, substitutedbenzyl ethers, silyl ethers, esters including sulfonic acid esters,carbonates, sulfates, and sulfonates. For example:

-   -   ethers (methyl, t-butyl, allyl);    -   substituted methyl ethers (methoxymethyl, methylthiomethyl,        t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl,        benzyloxymethyl, p-methoxybenzyloxymethyl,        (4-methoxyphenoxy)methyl, guaiacolmethyl, t-butoxymethyl,        4-pentenyloxymethyl, siloxymethyl, 2-methoxyethoxymethyl,        2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,        2-(trimethylsilyl)ethoxymethyl, tetrahydropyranyl,        3-bromotetrahydropyranyl, tetrahydropthiopyranyl,        1-methoxycyclohexyl, 4-methoxytetrahydropyranyl,        4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydropthiopyranyl        S,S-dioxido,        1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl,        1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,        2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl));    -   substituted ethyl ethers (1-ethoxyethyl,        1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,        1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,        2,2,2-trichloroethyl, 2-trimethylsilylethyl,        2-(phenylselenyl)ethyl,    -   p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl;    -   substituted benzyl ethers (p-methoxybenzyl, 3,4-dimethoxybenzyl,        o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl,        p-cyanobenzyl, p-phenylbenzyl, 2- and 4-picolyl,        3-methyl-2-picolyl N-oxido, diphenylmethyl,        p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl,        α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,        di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl,        4-(4′-bromophenacyloxy)phenyldiphenylmethyl,        4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,        4,4′,4″-tris(levulinoyloxyphenyl)methyl,        4,4′,4″-tris(benzoyloxyphenyl)methyl,        3-(imidazol-1-ylmethyl)bis(4′,4″-dimethoxyphenyl)methyl,        1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,        9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,        1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido);    -   silyl ethers (silyloxy groups) (trimethylsilyl, triethylsilyl,        triisopropylsilyl, dimethylisopropylsilyl,        diethylisopropylsilyl, dimethylthexylsilyl,        t-butyldimethylsilyl, t-butyldiphenylsilyl, tribenzylsilyl,        tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl,        t-butylmethoxyphenylsilyl);    -   esters (formate, benzoylformate, acetate, choroacetate,        dichloroacetate, trichloroacetate, trifluoroacetate,        methoxyacetate, triphenylmethoxyacetate, phenoxyacetate,        p-chlorophenoxyacetate, p-poly-phenylacetate,        3-phenylpropionate, 4-oxopentanoate (levulinate),        4,4-(ethylenedithio)pentanoate, pivaloate, adamantoate,        crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,        2,4,6-trimethylbenzoate (mesitoate));    -   carbonates (methyl, 9-fluorenylmethyl, ethyl,        2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,        2-(phenylsulfonyl)ethyl, 2-(triphenylphosphonio)ethyl, isobutyl,        vinyl, allyl, p-nitrophenyl, benzyl, p-methoxybenzyl,        3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, S-benzyl        thiocarbonate, 4-ethoxy-1-naphthyl, methyl dithiocarbonate);    -   groups with assisted cleavage (2-iodobenzoate, 4-azidobutyrate,        4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,        2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl carbonate,        4-(methylthiomethoxy)butyrate,        2-(methylthiomethoxymethyl)benzoate); miscellaneous esters        (2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3        tetramethylbutyl)phenoxyacetate,        2,4-bis(1,1-dimethylpropyl)phenoxyacetate,        chlorodiphenylacetate, isobutyrate, monosuccinate,        (E)-2-methyl-2-butenoate (tigloate),        o-(methoxycarbonyl)benzoate, p-poly-benzoate, α-naphthoate,        nitrate, alkyl N,N,N′,N′-tetramethyl-phosphorodiamidate,        n-phenylcarbamate, borate, dimethylphosphinothioyl,        2,4-dinitrophenylsulfenate); and    -   sulfonates (sulfate, methanesulfonate (mesylate),        benzylsulfonate, tosylate, triflate).

Uses of HDAC Inhibitors of the Invention

HDAC inhibitors of the invention may be used prophylactically or as atreatment for various neurodegenerative or neuromuscular conditions.More specifically, a HDAC inhibitor of the invention may be used todelay or prevent the onset of a neurodegenerative or neuromuscularcondition, as well as to treat a mammal suffering from aneurodegenerative or neuromuscular condition. Non-limiting examples ofneurodegenerative conditions include, without limitation, fragile Xsyndrome, Friedreich's ataxia, Huntington's disease, spinocerebellarataxias, amyotrophic lateral sclerosis, Kennedy's disease, spinal andbulbar muscular atrophy and Alzheimer's disease. Non-limiting examplesof neuromuscular conditions include spinal muscular atrophy and myotonicdystrophy.

Mammals, e.g. humans, to which HDAC inhibitors may be administeredinclude those suffering from the conditions discussed above as well asthose who are at risk for developing the above conditions. A mammal atrisk for developing a neurodegenerative condition may be identified innumerous ways, including, for example, first determining (1) the length,extent or number of repeats of particular nucleic acid sequences in theindividual's genome; the degree of acetylation of core histones; or theexpression level of a particular mRNA or protein, and then (2) comparingit with that of a normal individual. An individual at risk fordeveloping a neurodegenerative or neuromuscular condition is one who hasan aberrant number of repeat of a particular nucleic aid sequence,degree of acetylation of core histones or expression of a particulargene. For example, a mammal at risk for developing Friedreich's ataxiamay be identified by determining the length, extent or number of repeatsof a GAA triplet in the first intron of the fataxin gene. A mammal wouldbe at risk for Friedreich's ataxia if the above analysis indicates thatthere are more than 34 repeats of the GAA triplet, for example, if themammal has more than 66 repeats of the GAA triplet. A mammal at risk forFriedreich's ataxia could also be identified by determining the levelsof frataxin mRNA or protein expressed in the mammal. A mammal would beat risk for Friedreich's ataxia if the levels of frataxin mRNA orprotein is lower than the level normally observed in a healthyindividual such as for example, an unaffected sibling.

The amount of HDAC inhibitor to be administered to the mammal may be anyamount appropriate to restore the level of histone acetylation, or thelevel of mRNA or protein expression, in the afflicted mammal to thattypical of a healthy individual such as an unaffected sibling. Theamount of the HDAC inhibitor to be administered may be an effective doseor an appropriate fraction thereof. Such amounts will depend onindividual patient parameters including age, physical condition, size,weight, the condition being treated, the severity of the condition, andany concurrent treatment. For example, the effective dose range that isnecessary to prevent or delay the onset of the neurodegenerativecondition may be significantly lower than the effective dose range forinhibiting the progression of the condition being treated. Factors thatdetermine appropriate dosages are well known to those of ordinary skillin the art and may be addressed with routine experimentation. Forexample, determination of the physicochemical, toxicological andpharmacokinetic properties may be made using standard chemical andbiological assays and through the use of mathematical modelingtechniques known in the chemical, pharmacological and toxicologicalarts. The therapeutic utility and dosing regimen may be extrapolatedfrom the results of such techniques and through the use of appropriatepharmacokinetic and/or pharmacodynamic models. The precise amount ofHDAC inhibitor administered to a patient will be the responsibility ofthe attendant physician. However, that a patient may insist upon a lowerdose or tolerable dose for medical reasons, psychological reasons or forvirtually any other reasons.

HDAC inhibitors of the invention may be administered in numerous ways.For example, HDAC inhibitors of the invention may be administeredorally, rectally, topically, or by intramuscular, intraperitonealsubcutaneous or intravenous injection. Preferably, the inhibitors areadministered orally or by injection. Other routes include intrathecaladministration directly into spinal fluid and direct introduction onto,in the vicinity of or within the target cells. The route ofadministration may depend on the condition being treated and itsseverity.

HDAC inhibitors of the invention may be administered orally or byinjection at a dose of from 0.1 to 30 mg per kg weight of the mammal,preferably 2 to 15 mg/kg weight of the mammal. The dose range for adulthumans is generally from 8 to 2,400 mg/day and preferably 35 to 1,050mg/day. As certain HDAC inhibitors of the invention are long acting, itmay be advantageous to administer an initial dose of 70 to 2,400 mg thefirst day then a lower dose of 20 to 1,200 mg on subsequent days. If thesalt of the compound is administered, then the amount of saltadministered is calculated in terms of the base.

Pharmaceutical Compositions

HDAC inhibitors may be administered neat or, preferably, aspharmaceutical compositions. Pharmaceutical compositions of theinvention include an appropriate amount of the HDAC inhibitor incombination with an appropriate carrier as well as other usefulingredients.

HDAC inhibitors of the invention include the compounds of formula I, andwherein applicable, acceptable salts thereof. Acceptable salts include,but are not limited to, those prepared from the following acids: alkyl,alkenyl, aryl, alkylaryl and alkenylaryl mono-, di- and tricarboxylicacids of 1 to 20 carbon atoms, optionally substituted by 1 to 4hydroxyls; alkyl, alkenyl, aryl, alkylaryl and alkenylaryl mono-, di-and trisulfonic acids of 1 to 20 carbon atoms, optionally substituted by1 to 4 hydroxyls; and mineral acids. Examples include hydrochloric;hydrobromic; sulphuric; nitric; phosphoric; maleic; acetic; salicyclic;p-toluenesulfonic; tartaric; citric; methane sulphonic; formic; malonic;succinic; naphthalene-2-sulphonic; and benzenesulphonic acid. Also,pharmaceutically-acceptable salts may be prepared as amine salts,ammonium salts, or alkaline metal or alkaline earth salts, such assodium, potassium or calcium salts of the carboxylic acid group. Theseare formed from alkaline metal or alkaline earth metal bases or fromamine compounds. In addition, analogs of the foregoing compounds thatact as functional equivalents also are intended to be embraced asequivalents and within the scope of the invention.

Pharmaceutical compositions of HDAC inhibitors suitable for oraladministration may be in the form of (1) discrete units such ascapsules, cachets, tablets or lozenges each containing a predeterminedamount of the HDAC inhibitor; (2) a powder or granules; (3) a bolus,electuary or paste; (4) a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or (4) an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. Thus, compositions suitable for topicaladministration in the mouth, for example buccally or sublingually,include lozenges. Compositions suitable for parenteral administrationinclude aqueous and non-aqueous sterile suspensions or injectionsolutions. Compositions suitable for rectal administration may bepresented as a suppository.

Thus, pharmaceutical compositions of HDAC inhibitors may be formulatedusing a solid or liquid carrier. The solid or liquid carrier would becompatible with the other ingredients of the formulation and notdeleterious to the recipient. If the pharmaceutical composition is intablet form, then HDAC inhibitor is mixed with a carrier having thenecessary compression properties in suitable proportions and compactedin the shape and size desired. If the composition is in powder form, thecarrier is a finely divided solid in admixture with the finely dividedactive ingredient. The powders and tablets may contain up to 99% of theactive ingredient. Suitable solid carriers include, for example, calciumphosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins. A solidcarrier may include one or more substances that may act as flavoringagents, lubricants, solubilizers, suspending agents, fillers, glidants,compression aids, binders or tablet-disintegrating agents. A suitablecarrier may also be an encapsulating material.

If the composition is a solution, suspension, emulsion, syrup, elixirsor pressurized compositions, then liquid carriers may be used. In thiscase, the HDAC inhibitor is dissolved or suspended in a pharmaceuticallyacceptable liquid carrier. Suitable examples of liquid carriers for oraland parenteral administration include (1) water, (2) alcohols, e.g.monohydric alcohols and polyhydric alcohols such as glycols, and theirderivatives, and (3) oils, e.g. fractionated coconut oil and arachisoil. For parenteral administration, the carrier may also be an oilyester such as ethyl oleate and isopropyl myristate. Liquid carriers forpressurized compositions include halogenated hydrocarbon or otherpharmaceutically acceptable propellent. The liquid carrier may containother suitable pharmaceutical additives such as solubilizers;emulsifiers; buffers; preservatives; sweeteners; flavoring agents;suspending agents; thickening agents; colors; viscosity regulators;stabilizers; osmo-regulators; cellulose derivatives such as sodiumcarboxymethyl cellulose; anti-oxidants; and bacteriostats. Othercarriers include those used for formulating lozenges such as sucrose,acacia, tragacanth, gelatin and glycerin as well as those used informulating suppositories such as cocoa butter or polyethylene glycol.

If the composition is to be administered intravenously orintraperitoneally by infusion or injection, solutions of the HDACinhibitor may be prepared in water, optionally mixed with a nontoxicsurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms. The compositionsuitable for injection or infusion may include sterile aqueous solutionsor dispersions or sterile powders comprising the active ingredient,which are adapted for the extemporaneous preparation of sterileinjectable or infusible solutions or dispersions, optionallyencapsulated in liposomes. In all cases, the ultimate dosage form shouldbe sterile, fluid and stable under the conditions of manufacture andstorage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium as described above. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin. Sterileinjectable solutions are prepared by incorporating the HDAC inhibitor inthe required amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze-drying techniques, which yield a powder ofthe HDAC inhibitor, plus any additional desired ingredient present inthe previously sterile-filtered solutions.

Pharmaceutical compositions of the invention may be in unit-dose ormulti-dose form or in a form that allows for slow or controlled releaseof the HDAC inhibitor. Each unit-dose may be in the form of a tablet,capsule or packaged composition such as, for example, a packeted powder,vial, ampoule, prefilled syringe or sachet containing liquids. Theunit-dose form also may be the appropriate number of any suchcompositions in package form. Pharmaceutical compositions in multi-doseform may be in packaged in containers such as sealed ampoules and vials.In this case, the HDAC inhibitor may be stored in a freeze-dried(lyophilized) condition requiring only the addition of a sterile liquidcarrier immediately prior to use. In addition, extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Materials & Methods

Cell Culture

Epstein Barr virus transformed lymphoblast cell lines GM15850 from aFRDA patient (alleles with 650 and 1030 GAA repeats in the frataxingene, from the Coriell Cell Repository, Camden, N.J.), and GM15851 froman unaffected sibling (normal range of repeats), were propagated in RPMI1640 medium with 2 mM L-glutamine and 15% fetal bovine serum at 37° C.in 5% CO₂. Cell growth and morphology were monitored by phase contrastmicroscopy, and viability by trypan blue exclusion. HDAC inhibitors weredissolved in DMSO and added to the culture medium at the concentrationsindicated in the table and figure captions, for the indicated times. Thefinal DMSO concentration in the culture medium did not exceed 0.5%(v/v). All control samples were treated with the same concentration ofDMSO lacking compounds. The suppliers of the HDAC inhibitors were:valproic acid (VPA), Calbiochem (San Diego, Calif.); trichostatin A(TSA), suberoyl bis-hydroxamic acid (SBHA), suberoylanilide hydroxamicacid (SAHA), and BML-210, Bio-Mol (Plymouth Meeting, Pa.); each testedat the IC₅₀ value reported by the supplier, as indicated in the figures.

Real-Time Quantitative RT-PCR

Real-time quantitative RT-PCR analysis was performed essentially asdescribed in Chuma et al., Hepatology 37:198-207 (2003) using thefollowing primers for the frataxin gene: 5′-CAGAGGAAACGCTGGACTCT-3′(SEQID NO: 1) and 5′-AGCCAGATTTGCTTGTTTGG-3′ (SEQ ID NO:2). RNA wasstandardized by quantification of GAPDH mRNA as described in Pattyn etal., Nucl. Acids Res. 31:122-3 (2003), and all values are expressedrelative to GAPDH. Quantitative real-time RT-PCR was performed usingiScript One-Step RT-PCR kit with SYBR green (BioRad). Statisticalanalysis was performed on three independent quantitative RT-PCRexperiments for each RNA sample, and error bars shown in the figuresrepresent standard errors of the mean.

Western Blot Analysis

Protein levels in HDAC inhibitor-treated and untreated cells weremonitored by western blotting with antibodies to histones H3 and H4(Upstate Biotechnology) or with antibodies to the acetylated versions ofthese proteins. Histones were purified by acid extraction as describedin the protocols provided by Upstate Biotechnology. Antibodies to humanfrataxin were from Mitoscience (Eugene, Oreg.) and anti-actin antibodieswere from Santa Cruz Biotechnology (CA). Total cell extracts were usedfor frataxin and actin western blots. Signals were detected bychemiluminescence after probing the blot with HRP-conjugated secondaryantibody (Supersignal West, Pierce). To quantify the relative levels ofproteins, autoradiograms (within the linear response range of X-rayfilm) were converted into digital images and the signals quantifiedusing Molecular Dynamics ImageQuant software.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation was performed as previously described (seeLuo et al. Cell 92:463-73 (1998). For each immunoprecipitationexperiment, the amount of lysate corresponding to 25-50 μg of total DNAwas incubated with one of the following antibodies (each from UpsateBiotechnology, with the indicated catalogue numbers):anti-acetyl-Histone H3 (06-599), anti-acetyl-Histone H4 (06-598),anti-acetyl-Histone H3-Lys9 (07-352), anti-acetyl-Histone H3-Lys14(07-353), anti-acetyl-Histone H4-Lys5 (07-327), anti-acetyl-HistoneH4-Lys8 (07-328), anti-acetyl-Histone H4-Lys12 (07-595),anti-acetyl-Histone H4-Lys16 (07-329). Samples were quantified intriplicate by real time PCR, using the standard curve method, and errorbars shown in the figures represent standard errors of the mean. Theprimers used in this study were: for the frataxin promoter,5′-CCCCACATACCCAACTGCTG-3′ (SEQ ID NO:3) and 5′-GCCCGCCGCTTCTAAAATTC-3′(SEQ ID NO:4); for the region upstream of the GAA repeats in intron 1 ofthe frataxin gene, 5′-GAAACCCAAAGAATGGCTGTG-3′ (SEQ ID NO:5), and5′-TTCCCTCCTCGTGAAACACC-3′ (SEQ ID NO:6); for the region downstream ofthe GAA repeats in intron 1 of the frataxin gene,5′-CTGGAAAAATAGGCAAGTGTGG-3′ (SEQ ID NO:7) and5′-CAGGGGTGGAAGCCCAATAC-3′ (SEQ ID NO:8); and, for GAPDH,5′-CACCGTCAAGGCTGAGAACG-3′ (SEQ ID NO:9) and 5′-ATACCCAAGGGAGCCACACC-3′(SEQ ID NO:10).

Histone Deacetylase Assays

Each of the histone deacetylase inhibitors was assayed with the BioMolAK500 kit to determine IC₅₀ values. Samples were processed as describedby BioMol and read with a 96-well fluorescence plate reader. Asemi-logarithmic plot of the data was analyzed with Kaleidagraphsoftware to obtain the IC₅₀ value.

Human Subjects and Primary Lymphocytes

The Friedreich's Ataxia Research Alliance (Arlington, Va.) recruited aseries of families with affected individuals and siblings or parents foranonymous blood donation (with a Human Subjects Protocol approved by theScripps Clinic Human Subjects Committee and by NINDS, with appropriateinformed consent). Blood was collected in heparinized Vacutainer tubes(#364680, BD Biosciences) and lymphocytes were isolated by densitycentrifugation using Ficoll-Paque PLUS (Amersham Biosciences), accordingto the manufacturer. Lymphocytes were maintained in the same culturemedium and conditions as the established cell lines, and HDAC inhibitortreatment was as described above. Cells were treated with HDACinhibitors after 16 h, and RNA isolated after subsequent 48 hincubation. Under these culture conditions, no increases in cell numberwere observed.

Example 2—Synthesis of Bis-Amides HDAC Inhibitors by a Novel Two-StepProcedure

General Synthetic Procedure

Adipic acid 1a (n=3, Scheme 1), pimelic acid 1b (n=4) or suberic acid 1c(n=5) were used as the starting materials for the synthesis of the HDACinhibitors. The synthetic scheme is as shown below.

By reaction with acetic anhydride under reflux, the dicarboxylic acidsundergo intramolecular ring closure to compounds 2a, 2b and 2c. Incontrast to published results (Wong et al., J. Am. Chem. Soc. 125:5586-7(2003)), these anhydrides are further reacted without purification underring opening conditions with aniline to the precursor compounds 3a, 3band 3c in about 90% yield. Potent coupling conditions with1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (EDC) and1-hydroxy-7-azabenzotriazole (HOAt) produce a high conversion rate,resulting in fast reactions with high yields. By using these conditionsthe yield of 4b increased to 64% (compared to 33% (Wong et al., J. Am.Chem. Soc. 125:5586-7 (2003))). The yield of 4c (equivalent to BML-210)was 50-60% (overall). Comparable yields were obtained for each of thecompounds listed in Table 4.

The purity and identity of all compounds were verified by thin layerchromatography, analytical HPLC, MALDI-TOF MS, ¹³C- and ¹H-NMR. NMRspectra were recorded on Varian Mercury 300 or on DRX-500 from Bruker.¹³C spectra were measured using proton decoupling. All spectra werecalibrated to tetramethylsilane. An HPLC system was used for furtherpurification of some compounds (Hitachi L-6200A pump, L4200 UV-VISdetector, D-2500 chromato-integrator, and a Supelcosil PLC-18 (25cm×21.2 mm, 12 μm) column purchased from Supelco). The RP-HPLC systemwas set to a flow rate of 5 mL/min in 10% acetonitrile/water/0.1%TFA-100% acetonitrile/0.1% TFA for 0-60 minutes and then 100%acetonitrile/0.1% TFA 10% acetonitrile/water/0.1% TFA for 60-75 minutes.All MALDI-ToF spectra were measured on Voyager System 1089 from AppliedBiosystems; α-cyanohydroxy-cinnamic (CHCA) acid was used as matrix. Forflash column chromatography, silica gel (mesh 60-200) purchased fromJ.T. Baker was used. TLC plates were purchased from J.T. Baker (Si250F).

Abbreviations: CHCA (cyano-4-hydroxycinnamic acid), DCM(dichloromethane), diisopropylethylamine (DIPEA), dimethylsulfoxide(DMSO), dimethylformamide (DMF),N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC.HCl),1-hydroxy-1H-benzotriazole hydrate (HOBt), MeOH (methanol), roomtemperature (r.t), trifluoroacetic acid (TFA), and tetrahydrofurane(THF).

Detailed synthetic methods and analytical data for each compound are asfollows. The structures of the precursor compounds, PAOA, SAOA, andvarious derivatives are provided in the following Tables 1, 2 and 3.Each compound is an embodiment of the invention, by itself or incombination with other components and procedures described herein.

TABLE 1 Precursor Compounds Cmpd. No. Structure Mol. Wt. Formula P1

208.26 g/mol C₁₁H₁₆N₂O₂ 3a

221.25 g/mol C₁₂H₁₅NO₃ 3b

235.28 g/mol C₁₃H₁₇NO₃  3b2

236.27 g/mol C₁₂H₁₆N₂O₃  3b3

350.41 g/mol C₁₈H₂₆N₂O₅ 3c

249.31 g/mol C₁₄H₁₉NO₃

TABLE 2 HDAC Inhibitors Cmpd. No. Structure Mol. Wt. Formula 4a

311.38 g/mol C₁₈H₂₁N₃O₂ 4b (PAOA)

325.43 g/mol C₁₉H₂₃N₃O₂ 4c (SAOA) (BML-210)

339.43 g/mol C₂₀H₂₅N₃O₂ 5b

310.39 g/mol C₁₉H₂₂N₂O₂ 6b

325.43 g/mol C₁₉H₂₃N₃O₂ 6c

339.43 g/mol C₂₀H₂₅N₃O₂ 7b

325.43 g/mol C₁₉H₂₃N₃O₂ 7c

339.43 g/mol C₂₀H₂₅N₃O₂ 8b

311.38 g/mol C₁₈H₂₁N₃O₂ 8c

325.38 g/mol C₁₉H₂₃N₃O₂ 9b

340.42 g/mol C₂₀H₂₄N₂O₃ 9c

354.44 g/mol C₂₁H₂₆N₂O₃ 10b 

340.42 g/mol C₂₀H₂₄N₂O₃ 11b 

361.44 g/mol C₂₂H₂₃N₃O₂ 12b 

361.44 g/mol C₂₂H₂₃N₃O₂ 13b 

326.39 g/mol C₁₈H₂₂N₄O₂ 14b 

340.42 g/mol C₂₀H₂₄N₂O₃ 15b 

370.44 g/mol C₂₁H₂₆N₂O₄ 16b 

340.42 g/mol C₁₉H₂₄N₄O₂ 22a 

296.36 g/mol C₁₈H₂₀N₂O₂

Synthesis of the Precursor Compounds Compound P1:tert-Butyl-2-aminophenylcarbamate

A solution of Di-tert-butyldicarbonate (2.00 g, 10.0 mmol) in DMF (25mL) was added drop-wise to a stirred solution of 1,2-phenylendiamine(950 mg, 10.0 mmol) in 50 mL DMF at 55° C. After the addition thereaction mixture was stirred for 3 hours. The solvent was removed invacuo and the residue was taken up in ethylacetate (150 mL). The organicphase was washed three times with sat. NaCl solution (40 mL), dried overMgSO₄ and evaporated. The residue was recrystallized fromchloroform/n-hexane.

The reaction yielded 1.03 g (4.97 mmol, 49%) of P1 as a pale yellowsolid: TLC: R_(f)=0.49 (DCM/MeOH 20:1); ¹H-NMR (300 MHz, CDCl₃): δ =1.51(s, 9H), 6.35 (m, 1H), 6.72-6.78 (m, 2H), 6.98 (m, 1H), 7.25 (m, 1H).

Compound 3a: 6-Oxo-6-(phenylamino)hexanoic acid

A solution of adipic acid (5.00 g, 34.0 mmol) in acetic anhydride (10mL) was heated under reflux for 1 hour. After cooling to roomtemperature, the solvent was removed in vacuo. The crude yellow oil wasused without any further purification for the next step. Aniline (3.00mL, 28.7 mmol) was added to a stirred solution of the produced anhydridein anhydrous THF (10 mL). After stirring at room temperature for 1 hour,the solvent was removed and the residue was recrystallized fromwater/acetonitrile.

The reaction yielded 2.28 g (10.3 mmol, 36%) of 3a as a colorless solid:TLC: R_(f)=0.48 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=222.2(100, [M+H]⁺, calc. 222.2), 244.2 (20, [M+Na]⁺, calc. 244.2); ¹H-NMR(300 MHz, DMSO-D₆): δ =1.50-1.64 (m, 4H), 2.21-2.34 (m, 4H), 7.00-7.07(m, 2H), 7.02 (m, ³J=7.2 Hz, 1H), 7.28 (m, ³J=7.5 Hz, 2H), 7.59-7.62 (m,2H), 9.90 (s, 1H).

Compound 3b: 7-Oxo-7-(phenylamino)heptanoic acid

A solution of pimelic acid (5.00 g, 31.2 mmol) in acetic anhydride (10mL) was heated under reflux for 1 hour. After cooling to r.t. thesolvent was removed in vacuo. The crude yellow oil was used without anyfurther purification for the next step.

Aniline (3.00 mL, 28.7 mmol) was added to a stirred solution of theproduced anhydride in anhydrous THF (10 mL). After stirring at r.t. for30 minutes, the reaction mixture was diluted with water until acolorless solid precipitated, which was collected by filtration.Recrystallisation from water/acetonitrile gave the pure compound as acolorless solid.

The reaction yielded 3.52 g (15.0 mmol, 48%) of 3a as a colorless solid:TLC: R_(f)=0.68 (DCM/MeOH 9:1); MS (MALDI-ToF, CHCA): m/z (%)=236.3(100, [M+H]⁺, calc. 236.1), 258.4 (20, [M+Na]⁺, calc. 258.1); ¹H-NMR(500 MHz, DMSO-D₆): δ=1.28-1.34 (m, 2H), 1.49-1.62 (m, 4H), 1.55-1.58(m, 2H), 2.20 (t, ³J=9.0 Hz, 2H), 2.30 (t, ³J=9.0 Hz, 2H), 7.00 (m,³J=7.5 Hz, 1H), 7.26-7.29 (m, ³J=7.5 Hz, 2H), 7.57-7.59 (m, 2H), 9.83(s, 1H).

Compound 3b2: 7-Oxo-7-(pyridin-2-ylamino)heptanoic acid

A solution of pimelic acid (3.00 g, 18.7 mmol) in acetic anhydride (15mL) was heated under reflux for 1 hour. After cooling to r.t. thesolvent was removed in vacuo. The crude yellow oil was used without anyfurther purification for the next step.

2-Aminopyridine (1.75 g, 18.7 mmol) was added to a stirred solution ofthe produced anhydride in anhydrous THF (5 mL). After stifling thereaction for 2 hours at r.t., the solvent was removed in vacuo and theresidue was triturated with ethylacetate (200 mL). The organic phase waswashed with water, saturated NaCl solution and dried over anhydrousMgSO₄. After the solvent was removed, the residue was recrystallisedfrom water/trifluoroacetic acid (1:100) to give a colorless solid.

The reaction yielded 1.12 g (4.76 mmol, 25%) of 3b2 as a colorlesssolid: TLC: R_(f)=0.12 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z(%)=237.0 (100, [M+H]⁺, calc. 237.1); ¹H-NMR (300 MHz, methanol-D₄):δ=1.41-1.50 (m, 2H), 1.60-1.81 (m, 4H), 2.31 (t, ³J=7.2 Hz, 2H), 2.56(t, ³J=7.5 Hz, 2H), 7.45 (m, 1H), 7.64 (m, 1H), 8.21-8.28 (m, 1H),8.32-8.35 (m, 1H).

Compound 3b3: 7-(2-(tert-Butoxycarbonylamino)phenylamino)-7-oxoheptanoicacid

Pimelic acid (160 mg, 1.00 mmol) was refluxed in 5 mL acetic anhydridefor 1 hour. The solvent was removed to complete dryness. The residue wasdiluted in 10 mL dry THF, then P1 (200 mg, 0.96 mmol) in 5 mL dry THFwas added drop-wise. The reaction was stirred overnight and then dilutedwith water. The resulting colorless solid was collected by filtrationand recrystallized from ethanol.

The reaction yielded 275 mg (0.78 mmol, 82%) of 3b3 as a colorlesssolid: TLC: R_(f)=0.23 (DCM/MeOH 20:1); ¹H-NMR (500 MHz, CDCl₃): δ =1.50(s, 9H), 1.37-1.42 (m, 2H), 1.67-1.72 (m, 4H), 2.31-2.34 (m, 4H),7.00-7.07 (m, 2H), 7.13 (m, 1H), 7.36 (m, 1H), 8.25 (br. s, 1H).

Compound 3c: 8-Oxo-8-(phenylamino)octanoic acid

A solution of suberic acid (5.00 g, 28.7 mmol) in acetic anhydride (10mL) was heated under reflux for 1 hour. After cooling to r.t., thesolvent was removed in vacuo. The crude yellow oil was used without anyfurther purification for the next step.

Aniline (3.00 mL, 28.7 mmol) was added to a stirred solution of theproduced anhydride in anhydrous THF (10 mL). After stirring at r.t. for30 minutes, the reaction mixture was diluted with water until acolorless solid precipitated, which was collected by filtration.Recrystallisation from water/acetonitrile gave the pure compound as acolorless solid.

The reaction yielded 2.74 g (11.0 mmol, 39%) of 3c as a colorless solid:TLC: R_(f)=0.52 (DCM/MeOH 9:1); MS (MALDI-ToF, CHCA): m/z (%)=250.2(100, [M+H]⁺, calc. 250.1), 272.3 (26, [M+Na]⁺, calc. 272.1); ¹H-NMR(500 MHz, DMSO-D₆): δ=1.27-1.32 (m, 4H), 1.46-1.50 (m, 2H), 1.55-1.58(m, 2H), 2.18 (t, ³J=7.5 Hz, 2H), 2.28 (t, ³J=7.5 Hz, 2H), 6.99 (m,³J=7.5 Hz, 1H), 7.25-7.28 (m, ³J=7.5 Hz, 2H), 7.56-7.58 (m, ³J=7.5 Hz,2H), 9.82 (m, 1H), 11.9 (s, 1H).

Synthesis of Inhibitors Compound 4a:N¹-(2-Aminophenyl)-N⁷-phenylhexanediamide

A solution of P1 (86 mg, 0.40 mmol) in DMF (5 ml) was added drop-wise toa stirred solution of 3a (88 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41 mmol),HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5 mL).After stirring the reaction overnight, water was added until a colorlesssolid precipitated. The solid was collected by filtration andrecrystallized from water/acetonitrile. DCM/TFA (10 ml, 1:1) was addedto the dried solid and the solution was stirred for 2 h at r.t. Thesolvent was removed in vacuo and the residue lyophilized.

The reaction yielded 72 mg (0.17 mmol, 43%) of 4a as a colorless solid:TLC: R_(f)=0.69 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=312.3(100, [M+H]⁺, calc. 312.2), 334.4 (2, [M+Na]⁺, calc. 334.2); ¹H-NMR (300MHz, DMSO-D₆): δ =1.60-1.67 (m, 4H), 2.32-2.40 (m, 4H), 6.90-7.12 (m,4H), 7.23-7.30 (m, 3H), 7.58-7.60 (m, 2H), 9.59 (s, 1H), 9.91 (s, 1H).

Compound 4b: N¹-(2-Aminophenyl)-N⁷-phenylheptanediamide

A solution of compound 3b (94 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5mL) was added dropwise to a stirred and cooled solution of1,2-phenylenediamine (432 mg, 4.00 mmol) in DMF (3 mL). After stiflingthe reaction overnight, water was added until a colorless solidprecipitated. The solid was collected by filtration and recrystallisedfrom water/ethanol. The product is further purified by preparativeRP-HPLC.

The reaction yielded 60 mg (0.17 mmol, 43%) of 4b as a colorless solid:RP-HPLC: R_(t)=32 minutes; TLC: R_(f)=0.46 (DCM/MeOH 20:1); MS(MALDI-ToF, CHCA): m/z (%)=326.2 (100, [M+H]⁺, calc. 326.2), 348.2 (40,[M+Na]⁺, calc. 348.2); ¹H-NMR (300 MHz, DMSO-D₆): δ=1.34-1.38 (m, 2H),1.58-1.69 (m, 4H), 2.29-2.39 (m, 4H), 6.95-7.15 (m, 4H), 7.23-7.30 (m,3H), 7.54-7.60 (m, 2H), 9.68 (s, 1H), 9.89 (s, 1H).

Compound 4c: N¹-(2-Aminophenyl)-N⁸-phenyloctanediamide

A solution of compound 3c (100 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5mL) was added dropwise to a stirred and cooled solution of1,2-phenylenediamine (432 mg, 4.00 mmol) in DMF (3 mL). After stiflingthe reaction overnight, water was added until a colorless solidprecipitated. The solid was collected by filtration and recrystallisedfrom water/ethanol.

The reaction yielded 60 mg (0.17 mmol, 43%) of 4c as a colorless solid:TLC: R_(f)=0.63 (DCM/MeOH 9:1); MS (MALDI-ToF, CHCA): m/z (%)=340.2(100, [M+H]⁺, calc. 340.2), 362.2 (15, [M+Na]⁺, calc. 362.2); ¹H-NMR(300 MHz, DMSO-D₆): δ=1.31-1.32 (m, 4H), 1.56-1.62 (m, 4H), 2.26-2.31(m, 4H), 6.95-7.15 (m, 4H), 7.22-7.29 (m, 3H), 7.53-7.61 (m, 2H), 9.68(s, 1H), 9.89 (s, 1H).

Compound 5b: N¹-Phenyl-N⁷-phenylheptanediamide

A solution of compound 3b (94 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5mL) was added dropwise to a stirred and cooled solution of aniline (37mg, 0.40 mmol) in DMF (3 mL). After stirring the reaction overnight,water was added to the reaction mixture until a colorless solidprecipitated. The crude product was collected by filtration andrecrystallised from water/ethanol.

The reaction yielded 52 mg (0.17 mmol, 42%) of 5b as a colorless solid:TLC: R_(f)=0.46 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=311.1(100, [M+H]⁺, calc. 311.2), 333.1 (90, [M+Na]⁺, calc. 333.3); ¹H-NMR(500 MHz, DMSO-D₆): δ=1.32-1.35 (m, 2H), 1.59-1.64 (m, 4H), 2.31 (t,³J=7.3 Hz, 4H), 7.00 (m, ³J=7.3 Hz, 2H), 7.27 (m, ³J=7.3 Hz, 4H),7.57-7.59 (m, 4H), 9.86 (s, 2H).

Compound 6b: N¹-(3-Aminophenyl)-N⁷-phenylheptanediamide

A solution of compound 3b (94 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5mL) was added dropwise to a stirred and cooled solution of1,3-phenylenediamine (432 mg, 4.00 mmol) in DMF (3 mL). After stiflingthe reaction overnight, the solvent was removed in vacuo. The residuewas triturated with ethylacetate/water. The phases were separated andthe aqueous phase was extracted two times with ethylacetate (50 mL). Thecombined organic phases were washed one time with saturated NaClsolution and dried over MgSO₄. After evaporation of the solvent, theresidue was further purified by flash-chromatography(dichloro-methane/methanol 80:1).

The reaction yielded 24 mg (0.06 mmol, 15%) of 6b as a colorless solid:TLC: R_(f)=0.61 (DCM/MeOH 9:1); MS (MALDI-ToF, CHCA): m/z (%)=326.3(100, [M+H]⁺, calc. 326.2), 348.4 (5, [M+Na]⁺, calc. 348.2); ¹H-NMR (300MHz, DMSO-D₆): δ=1.24-1.32 (m, 2H), 1.50-1.60 (m, 4H), 2.24 (t, ³J=7.5Hz, 2H), 2.25 (t, ³J=7.5 Hz, 2H), 6.68 (m, 1H), 6.94 (m, 1H), 7.11-7.23(m, 4H), 7.50-7.53 (m, 3H), 9.81 (s, 1H), 9.92 (s, 1H).

Compound 6c: N¹-(3-Aminophenyl)-N⁸-phenyloctanediamide

A solution of compound 3c (100 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5mL) was added dropwise to a stirred and cooled solution of1,3-phenylenediamine (432 mg, 4.00 mmol) in DMF (3 mL). After stiflingthe reaction overnight, water was added until a colorless solidprecipitated. The solid was collected by filtration and recrystallisedfrom water/ethanol. The product was further purified by RP-HPLC.

The reaction yielded 52 mg (0.15 mmol, 38%) of 6c as a colorless solid:RP-HPLC: R_(t)=36 minutes; TLC: R_(f)=0.66 (DCM/MeOH 9:1); MS(MALDI-ToF, CHCA): m/z (%)=340.3 (100, [M+H]⁺, calc. 340.2), 362.3 (30,[M+Na]⁺, calc. 362.2); ¹H-NMR (300 MHz, DMSO-D₆): δ=1.32-1.33 (m, 4H),1.51-1.65 (m, 4H), 2.26-2.31 (m, 4H), 6.60 (m, 1H), 6.98-7.06 (m, 2H),7.11-7.17 (m, 1H), 7.23-7.30 (m, 2H), 7.41 (m, 1H), 7.56-7.59 (m, 2H),9.86 (s, 1H), 9.87 (s, 1H).

Compound 7b: N¹-(4-Aminophenyl)-N⁷-phenylheptanediamide

A solution of compound 3b (94 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5mL) was added dropwise to a stirred and cooled solution of1,4-phenylenediamine (432 mg, 4.00 mmol) in DMF (3 mL). After stiflingthe reaction overnight, the solvent was removed in vacuo. The residuewas triturated with ethylacetate/water. The phases were separated andthe aqueous phase was extracted two times with ethylacetate (50 mL). Thecombined organic phases were washed one time with saturated NaClsolution and dried over MgSO₄. After evaporation of the solvent, theresidue is recrystallised from water/acetonitrile. The compound was thenfurther purified by RP-HPLC.

The reaction yielded 25 mg (0.06 mmol, 15%) of 7b as a colorless solid:RP-HPLC: R_(t)=30 minutes; TLC: R_(f)=0.38 (DCM/MeOH 9:1); MS(MALDI-ToF, CHCA): m/z (%)=326.4 (100, [M+H]⁺, calc. 326.2), 348.3 (50,[M+Na]⁺, calc. 348.2); ¹H-NMR (300 MHz, DMSO-D₆): δ=1.27-1.37 (m, 2H),1.56-1.66 (m, 4H), 2.24 (m, 4H), 6.98-7.03 (m, 1H), 7.12-7.17 (m, 2H),7.24-7.29 (m, 2H), 7.56-7.63 (m, 4H), 9.87 (s, 1H), 9.98 (s, 1H).

Compound 7c: N¹-(4-Aminophenyl)-N⁸-phenyloctanediamide

A solution of compound 3c (100 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5mL) was added dropwise to a stirred and cooled solution of1,4-phenylenediamine (432 mg, 4.00 mmol) in DMF (3 mL). After stirringthe reaction overnight, the solvent was removed in vacuo and the slurrywas pardoned between water and ethylacetate. The phases were separatedand the aqueous phase was extracted two more times with ethylacetate (20mL). The aqueous phase is then concentrated in vacuo and treated withacetonitrile/water (1:4) until a colorless solid precipitated. Theproduct is isolated by filtration and purified by RP-HPLC.

The reaction yielded 25 mg (0.07 mmol, 16%) of 7c as a colorless solid:RP-HPLC: R_(t)=31 minutes; TLC: R_(f)=0.66 (DCM/MeOH 9:1); MS(MALDI-ToF, CHCA): m/z (%)=340.5 (80, [M+H]⁺, calc. 340.2), 362.4 (15,[M+Na]⁺, calc. 362.2); ¹H-NMR (300 MHz, DMSO-D₆): δ=1.25-1.39 (m, 4H),1.54-1.67 (m, 4H), 2.27 (m, 4H), 7.00-7.09 (m, 1H), 7.14-7.20 (m, 2H),7.28-7.34 (m, 2H), 7.54-7.65 (m, 4H), 9.78 (s, 1H), 9.95 (s, 1H).

Compound 8b: N¹-Phenyl-N⁷-(pyridin-2-yl)heptanediamide

A solution of 2-aminopyridine (40 mg, 0.40 mmol) in DMF (5 mL) was addeddropwise to a cooled solution of compound 3b (94 mg, 0.40 mmol), EDC.HCl(78 mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol)in DMF (5 mL). After stifling overnight, water was added to the reactionmixture until a colorless solid precipitated. The product was collectedby filtration and recrystallised from water/acetonitrile.

The reaction yielded 80 mg (0.25 mmol, 64%) of 8b as a colorless solid:TLC: R_(f)=0.49 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=312.3(100, [M+H]⁺, calc. 312.2), 334.3 (18, [M+Na]⁺, calc. 334.2); ¹H-NMR(300 MHz, DMSO-D₆): δ=1.29-1.38 (m, 2H), 1.59-1.64 (m, 4H), 2.30 (t,³J=7.5 Hz, 2H), 2.43 (t, ³J=7.2 Hz, 2H), 7.00 (m, ³J=7.2 Hz, 1H), 7.17(m, 1H) 7.24-7.29 (m, ³J=7.5 Hz, 2H), 7.56-7.59 (m, ³J=7.8 Hz, 2H),7.85-7.98 (m, 2H), 8.30 (m, 1H), 9.86 (s, 1H), 10.8 (s, 1H).

Compound 8c: N¹-Phenyl-N⁸-(pyridin-2-yl)octanediamide

A solution of 2-aminopyridine (40 mg, 0.40 mmol) in DMF (5 mL) was addeddropwise to a cooled solution of compound 3c (100 mg, 0.40 mmol),EDC.HCl (78 mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl,0.40 mmol) in DMF (5 mL). After stifling overnight, water was added tothe reaction mixture until a colorless solid precipitated. The productwas collected by filtration and recrystallised from water/acetonitrile.

The reaction yielded 23 mg (0.07 mmol, 18%) of 8c as a colorless solid:TLC: R_(f)=0.25 (DCM/MeOH 9:1); MS (MALDI-ToF, CHCA): m/z (%)=326.1(100, [M+H]⁺, calc. 326.2), 348.1 (18, [M+Na]⁺, calc. 348.2); ¹H-NMR(300 MHz, methanol-D₄): δ=1.39-1.46 (m, 4H), 1.59-1.78 (m, 4H), 2.28 (t,³J=7.5 Hz, 2H), 2.54 (t, ³J=7.5 Hz, 2H), 7.06 (m, 1H), 7.25-7.30 (m, 2H)7.42-7.47 (m, 1H), 7.51-7.54 (m, 2H), 7.60 (m, 1H), 8.23 (m, 1H), 8.31(m, 1H).

Compound 9b: N¹-(2-Methoxyphenyl)-N⁷-phenylheptanediamide

A solution of 2-methoxyaniline (50 mg, 0.40 mmol) in DMF (3 mL) wasadded dropwise to a cooled solution of compound 3b (94 mg, 0.40 mmol),EDC.HCl (78 mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl,0.40 mmol) in DMF (5 mL). After stifling overnight, water was added tothe reaction mixture until a colorless solid precipitated. The crudeproduct was collected by filtration and recrystallised fromwater/acetonitrile.

The reaction yielded 95 mg (0.28 mmol, 70%) of 9b as a colorless solid:TLC: R_(f)=0.27 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=341.4(100, [M+H]⁺, calc. 341.2), 363.4 (35, [M+Na]⁺, calc. 363.2); ¹H-NMR(300 MHz, DMSO-D₆): δ=1.29-1.38 (m, 2H), 1.55-1.67 (m, 4H), 2.30 (t,³J=7.4 Hz, 2H), 2.38 (t, ³J=7.4 Hz, 2H), 3.80 (s, 3H), 6.85 (m, 1H),6.98-7.07 (m, 2H), 7.25-7.30 (m, ³J=7.5 Hz, 2H), 7.58-7.60 (m, 2H), 7.93(m, 1H), 9.03 (s, 1H), 9.86 (s, 1H).

Compound 9c: N¹-(2-Methoxyphenyl)-N⁸-phenyloctanediamide

A solution of 2-methoxyaniline (50 mg, 0.40 mmol) in DMF (3 mL) wasadded dropwise to a cooled solution of compound 3c (100 mg, 0.40 mmol),EDC.HCl (78 mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl,0.40 mmol) in DMF (5 mL). After stifling overnight, water was added tothe reaction mixture until a colorless solid precipitated. The crudeproduct was collected by filtration and recrystallised fromwater/acetonitrile.

The reaction yielded 58 mg (0.16 mmol, 41%) of 9c as a colorless solid:TLC: R_(f)=0.61 (DCM/MeOH 9:1); MS (MALDI-ToF, CHCA): m/z (%)=355.4(100, [M+H]⁺, calc. 355.2), 377.3 (18, [M+Na]⁺, calc. 377.2); ¹H-NMR(300 MHz, DMSO-D₆): δ=1.30-1.36 (m, 4H), 1.50-1.68 (m, 4H), 2.30-2.39(m, 4H), 3.81 (s, 3H), 6.88 (m, 1H), 6.90-7.10 (m, 3H), 7.26-7.30 (m,2H), 7.58-7.61 (m, 2H), 7.93 (m, 1H), 9.01 (s, 1H), 9.86 (s, 1H).

Compound 10b: N¹-(4-Methoxyphenyl)-N⁷-phenylheptanediamide

A solution of 4-methoxyaniline (50 mg, 0.40 mmol) in DMF (5 mL) wasadded dropwise to a cooled solution of compound 3b (94 mg, 0.40 mmol),EDC.HCl (78 mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl,0.40 mmol) in DMF (5 mL). After stifling overnight, water was added tothe reaction mixture until a colorless solid precipitated. The crudeproduct was collected by filtration and recrystallised fromwater/acetonitrile.

The reaction yielded 63 mg (0.18 mmol, 46%) of 10b as a colorless solid:TLC: R_(f)=0.38 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=341.4(100, [M+H]⁺, calc. 341.2), 363.5 (15, [M+Na]⁺, calc. 363.2); ¹H-NMR(300 MHz, DMSO-D₆): δ=1.25-1.42 (m, 2H), 1.55-1.73 (m, 4H), 2.27-2.32(m, 4H), 3.70 (s, 3H), 6.84-6.86 (m, 2H), 7.01 (m, 1H), 7.25-7.30 (m,2H), 7.48-7.60 (m, 4H), 9.72 (s, 1H), 9.86 (s, 1H).

Compound 11b: N¹-Phenyl-N⁷-(quinolin-8-yl)heptanediamide

A solution of 8-aminoquinoline (58 mg, 0.40 mmol) in 3 mL DMF was addeddropwise to a stirred and cooled solution of compound 3b (95 mg, 0.40mmol), EDC.HCl (78 mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68μl, 0.40 mmol) in DMF (5 mL). After stirring the reaction overnight, thereaction mixture was diluted with water until a colorless solidprecipitated. The solid was collected by filtration, washed with waterand recrystallised from water/acetonintrile.

The reaction yielded 34 mg (0.10 mmol, 25%) of 11b as a colorless solid:TLC: R_(f)=0.47 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=362.8(100, [M+H]⁺, calc. 362.2), 384.8 (10, [M+Na]⁺, calc. 384.2); ¹H-NMR(300 MHz, DMSO-D₆): δ=1.36-1.44 (m, 2H), 1.59-1.73 (m, 4H), 2.32 (t,³J=7.4 Hz, 2H), 2.59 (t, ³J=7.4 Hz, 2H), 7.00 (m, 1H), 7.23-7.29 (m,2H), 7.54-7.68 (m, 5H), 8.41 (m, 1H), 8.62 (m, 1H), 8.92 (m, 1H), 9.86(s, 1H), 10.01 (s, 1H).

Compound 12b: N¹-Phenyl-N⁷-(quinolin-3-yl)heptanediamide

A solution of 3-aminoquinoline (58 mg, 0.40 mmol) in 3 mL DMF was addeddropwise to a stirred and cooled solution of compound 3b (95 mg, 0.40mmol), EDC.HCl (78 mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68μl, 0.40 mmol) in DMF (5 mL). After stifling the reaction overnight, thereaction mixture was diluted with water until a colorless solidprecipitated. The solid was collected by filtration, washed with waterand recrystallised from water/acetonitrile.

The reaction yielded 47 mg (0.13 mmol, 32%) of 12b as a colorless solid:TLC: R_(f)=0.47 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=362.7(100, [M+H]⁺, calc. 362.2), 384.6 (20, [M+Na]⁺, calc. 384.2); ¹H-NMR(300 MHz, DMSO-D₆): δ=1.35-1.45 (m, 2H), 1.61-1.75 (m, 4H), 2.34 (t,³J=7.2 Hz, 2H), 2.45 (t, ³J=7.5 Hz, 2H), 7.02 (m, 1H), 7.25-7.30 (m,2H), 7.58-7.61 (m, 2H), 7.67 (m, 1H), 7.76 (m, 1H), 8.02-8.05 (m, 2H),8.88 (m, 1H), 9.10 (m, 1H), 9.89 (s, 1H), 10.60 (s, 1H).

Compound 13b: N¹-(2-Aminophenyl)-N⁷-(pyridin-2-yl)heptanediamide

A solution of compound 3b2 (95 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5mL) was added dropwise to a stirred and cooled solution of1,2-phenylenediamine (432 mg, 4.00 mmol) in DMF (3 mL). After stiflingthe reaction overnight, the solvent was removed and the residue wasdirectly purified by flash-column chromatography(dichloromethane/methanol 80:1-40:1).

The reaction yielded 86 mg (0.27 mmol, 67%) of 13b as a colorless oil:TLC: R_(f)=0.37 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=327.4(100, [M+H]⁺, calc. 327.2), 349.5 (5, [M+Na]⁺, calc. 349.2); ¹H-NMR (300MHz, DMSO-D₆): δ=1.24-1.38 (m, 2H), 1.55-1.73 (m, 4H), 2.18-2.35 (m,4H), 6.84-6.95 (m, 2H), 7.28-7.40 (m, 3H), 7.65 (m, 1H), 8.25 (m, 1H),8.34 (m, 1H), 9.72 (s, 1H), 10.36 (s, 1H).

Compound 14b: N¹-(3-Methoxyphenyl)-N⁷-phenylheptanediamide

A solution of 3-methoxyaniline (50 mg, 0.40 mmol) in DMF (3 mL) wasadded dropwise to a cooled solution of compound 3b (95 mg, 0.40 mmol),EDC.HCl (78 mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl,0.40 mmol) in DMF (5 mL). After stifling overnight, the solvent wasremoved in vacuo and the residue was triturated with ethylacetate/water(150 mL/20 mL). The phases were separated and the organic phase waswashed two times with saturated NaCl solution, dried over MgSO₄ andevaporated. The residue was purified by column chromatography(dichloromethane/methanol 20:1).

The reaction yielded 84 mg (0.24 mmol, 62%) of 14b as a colorless oil:TLC: R_(f)=0.32 (DCM/MeOH 9:1); MS (MALDI-ToF, CHCA): m/z (%)=341.2 (60,[M+H]⁺, calc. 341.2), 363.2 (70, [M+Na]⁺, calc. 363.2); ¹H-NMR (500 MHz,CDCl₃): δ=1.38-1.42 (m, 2H), 1.69-1.72 (m, 4H), 2.31-2.35 (m, 4H), 3.74(s, 3H), 6.63 (m, 1H), 7.00 (m, 1H), 7.06 (m, 1H), 7.10 (m, 1H), 7.15(m, 1H), 7.24-7.27 (m, 2H), 7.30-7.51 (m, 2H), 7.82-7.86 (m, 2H).

Compound 15b: N¹-(2,4-Dimethoxyphenyl)-N⁷-phenylheptanediamide

A solution of 2,4-dimethoxyaniline (48 mg, 0.40 mmol) in DMF (3 mL) wasadded dropwise to a cooled solution of compound 3b (95 mg, 0.40 mmol),EDC.HCl (78 mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μl,0.40 mmol) in DMF (5 mL). After stirring the reaction overnight, thereaction mixture was diluted with water until a colorless solidprecipitated. The solid was collected by filtration, washed with waterand recrystallised from water/acetonitrile.

The reaction yielded 97 mg (0.26 mmol, 65%) of 15b as a colorless oil:TLC: R_(f)=0.36 (DCM/MeOH 9:1); MS (MALDI-ToF, CHCA): m/z (%)=371.5(100, [M+H]⁺, calc. 371.2), 393.5 (50, [M+Na]⁺, calc. 393.2); ¹H-NMR(500 MHz, CDCl₃): δ=1.42-1.48 (m, 2H), 1.73-1.79 (m, 4H), 2.34-2.40 (m,4H), 3.77 (s, 3H), 3.82 (s, 3H), 6.42-6.46 (m, 2H), 7.06 (m, 1H),7.26-7.29 (m, 2H), 7.51-7.56 (m, 3H), 7.70 (m, 1H), 8.16 (m, 1H).

Compound 16b: N¹,N⁷-Bis(2-aminophenyl)heptanediamide

A solution of 3b3 (140 mg, 0.40 mmol), EDC.HCl (78 mg, 0.41 mmol), HOBt(64 mg, 0.40 mmol) and DIPEA (68 μl, 0.40 mmol) in DMF (5 mL) was addeddrop-wise to a stirred and cooled solution of P1 (82 mg, 0.40 mmol) inDMF (3 mL). After stirring the reaction overnight, the solvent wasremoved and the residue was triturated with ethyl-acetate/water (150 mL,2:1). The phases were separated and the organic phase was washed twotimes with 5% cold citric acid (20 mL), two times with sat. NaHCO₃solution (20 mL), two times with saturated NaCl solution (40 mL), driedover MgSO₄ and evaporated. The residue was purified byflash-chromatography (DCM/MeOH 40:1).

The reaction yielded 186 mg (0.34 mg, 86%) of a colorless solid: TLC:R_(f)=0.21 (DCM/MeOH 20:1).

The Boc protecting groups were cleaved by adding a mixture of DCM/TFA (5mL, 3:2) and stirring for 2 hours at room temperature. After the solventwas removed, the product was lyophilized. If necessary the product canbe further purified by preparative HPLC.

This reaction yielded 185 mg (0.32 mmol, 80%) of a colorless solid:RP-HPLC: R_(t)=27.0 min; TLC: R_(f)=0.05 (DCM/MeOH 20:1); MS (MALDI-ToF,CHCA): m/z (%)=341.2 (100, [M+H]⁺, calc. 341.2), 363.2 (20, [M+Na]⁺,calc. 363.2); ¹H-NMR (300 MHz, DMSO-D₆): δ =1.38-1.45 (m, 2H), 1.63-1.71(m, 4H), 2.39-2.43 (m, 4H), 7.18-7.34 (m, 8H), 9.59-9.85 (br. s, 4H),9.97 (s, 2H).

Compound 22a: N¹-Phenyl-N⁷-phenylhexanediamide

A solution of aniline (37 mg, 0.40 mmol) in DMF (5 mL) was addeddrop-wise to a stirred solution of 3a (88 mg, 0.40 mmol), EDC.HCl (78mg, 0.41 mmol), HOBt (64 mg, 0.40 mmol) and DIPEA (68 μL, 0.40 mmol) inDMF (5 mL). After stirring the reaction overnight, water was added untila colorless solid precipitated. The crude product was collected byfiltration and recrystallized from water/ethanol.

This reaction yielded 64 mg (0.21 mmol, 54%) of 22a as a colorlesssolid: TLC: R_(f)=0.35 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z(%)=297.4 (80, [M+H]⁺, calc. 297.2), 319.4 (20, [M+Na]⁺, calc. 319.2);¹H-NMR (300 MHz, DMSO-D₆): δ =1.60-1.67 (m, 4H), 2.33-2.41 (m, 4H),6.99-7.03 (m, ³J=7.0 Hz, 2H), 7.26-7.30 (m, ³J=7.6 Hz, 4H), 7.58-7.60(m, ³J=7.6 Hz, 4H), 9.88 (s, 2H).

Synthesis of Additional Precursors Compound P2:tert-Butyl-4-aminophenylcarbamate

A solution of di-tert-butyldicarbonate (2.0 g, 10 mmol) in DMF (25 mL)was added dropwise to stirred solution of 1,4-phenylenediamine (1.08 g,10 mmol) in 50 mL DMF at 55° C. After the addition the reaction mixturewas stirred for 2 hours. The solvent was removed in vacuo and theresidue was taken up in ethylacetate (150 mL). The organic phase waswashed three times with 5% citric acid, three times with saturatedsodium-bicarbonate and three times with saturated NaCl solution (40 mL).The organic phase was then dried over MgSO₄ and evaporated. The residuewas recrystallised from ethylacetate.

This reaction yielded 1.35 g (6.50 mmol, 65%) of P2 as a colorlesssolid: TLC: R_(f)=0.39 (DCM/MeOH 20:1); ¹H-NMR (500 MHz, CDCl₃): δ 1.50(s, 9H), 3.50 (br. s, 2H), 6.33 (br. s, 1H), 6.60-6.64 (AA′BB′, 2H),7.11-7.13 (AA′BB′, 2H). ¹³C-NMR (128 MHz, CDCl₃): δ 28.4, 80.0, 115.6,116.7, 120.9, 129.7, 153.3.

Compound P3: 7-(4-(tert-Butoxycarbonylamino)phenylamino)-7-oxoheptanoicacid

Pimelic acid (200 mg, 1.25 mmol) was refluxed in 10 mL acetic anhydridefor 30 minutes. The solvent was removed to complete dryness. The residuewas diluted in 5 mL dry THF, then P2 (208 mg, 1.00 mmol) in 5 mL dry THFwas added dropwise. The reaction was stirred overnight and then thesolvent was evaporated. The residue was taken up in 100 mL ethylacetateand then washed two times with 50 mL with water and two times withsaturated NaCl. After drying over MgSO₄ the solvent was evaporated. Thecrude residue was purified by flashcolumn chromatography on silica gel(DCM/MeOH 40:1).

This reaction yielded 140 mg (0.4 mmol, 40%) of P3 as a colorless solid:TLC: R_(f)=0.14 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=351.3(15, [M+H]⁺, calc. 351.2), 373.4 (100, [M+Na]⁺, calc. 373.2); ¹H-NMR(500 MHz, D₆-DMSO): δ1.24-1.29 (m, 4H), 1.45 (s, 9H), 1.47-1.53 (m, 4H),2.21-2.26 (m, 2H), 7.33 (AA′BB′, 2H), 7.44 (AA′BB′, 2H), 9.20 (s, 1H),9.70 (s, 1H).

Compound 80b: 7-Oxo-7-(2-methoxyphenylamino)heptanoic acid

A solution of pimelic acid (5.00 g, 31.2 mmol) in acetic anhydride (10mL) was heated under reflux for 1 hour. After cooling to roomtemperature, the solvent was removed in vacuo. The crude yellow oil wasused without any further purification for the next step. o-Anisidine(2.10 mL, 28.7 mmol) was added to a stirred solution of the producedanhydride in anhydrous THF (10 mL). After stirring at r.t. for 30 min,the reaction mixture was diluted with water until a colorless solidprecipitated, which was collected by filtration. Recrystallisation fromwater/acetonitrile gave the pure compound as a colorless solid.

This reaction yielded 1.86 g (7.02 mmol, 25%) of 80b as a colorlesssolid: TLC: R_(f)=0.21 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z(%)=266.3 (100, [M+H]⁺, calc. 266.1), 288.4 (24, [M+Na]⁺, calc. 288.1);¹H-NMR (500 MHz, D₄-MeOH): δ 1.31-1.36 (m, 2H), 1.58-1.64 (m, 4H),2.28-2.41 (m, 4H), 3.86 (s, 3H), 6.91-7.02 (m, 1H), 7.07-7.12 (m, 2H),7.94 (m, 1H).

Compound 100b: 7-Oxo-7-(o-tolylamino)heptanoic acid

A solution of pimelic acid (2.50 g, 16.4 mmol) in acetic anhydride (10mL) was heated under reflux for 20 minutes. After cooling to roomtemperature, the solvent was removed in vacuo. The crude yellow oil wasused without any further purification for the next step. o-Toluidine(1.77 mL, 16.0 mmol) was added to a stirred solution of the producedanhydride in anhydrous THF (10 mL). After stifling at room temperaturefor 1 hour, the reaction mixture was diluted with water until acolorless solid precipitated, which was collected by filtration.Recrystallisation from water/ethanol gave the pure compound as acolorless solid.

This reaction yielded 1.26 g (5.1 mmol, 31%) of 100b as a colorlesssolid: TLC: R_(f)=0.16 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z(%)=250.3 (73, [M+H]⁺, calc. 250.1), 272.4 (56, [M+Na]⁺, calc. 272.1);¹H-NMR (500 MHz, D₄-MeOH): δ 1.41-1.49 (m, 2H), 1.64-1.81 (m, 4H), 2.23(s, 3H) 2.30 (d, ³J=7.6 Hz, 2H), 2.41 (d, ³J=7.6 Hz, 2H), 3.32 (s, 3H),7.01-7.18 (m, 2H), 7.21-7.23 (m, 1H), 7.25-7.29 (m, 1H).

Compound 101b: 7-Oxo-7-(p-tolylamino)heptanoic acid

The synthesis method was the same as that for 100b, with the exceptionthat p-toluidine was used in place of o-toluidine.

This reaction yielded 1.56 g (6.3 mmol, 38%) of 101b as a colorlesssolid: TLC: R_(f)=0.17 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z(%)=250.3 (100, [M+H]⁺, calc. 250.1), 272.4 (24, [M+Na]⁺, calc. 272.1);¹H-NMR (500 MHz, D₄-MeOH): δ 1.42-1.50 (m, 2H), 1.63-1.79 (m, 4H), 2.35(s, 3H) 2.31 (d, ³J=7.5 Hz, 2H), 2.43 (d, ³J=7.6 Hz, 2H), 3.32 (s, 3H),7.09-7.12 (AA′BB′, 2H), 7.48-7.51 (AA′BB′, 2H).

Synthesis of Additional Inhibitors

TABLE 3 Additional Inhibitors Number Structure Mol. Wt. Formula 81b

355.43 g/mol C₂₀H₂₅N₃O₃ 82b

355.43 g/mol C₂₀H₂₅N₃O₃ 102b 

339.43 g/mol C₂₀H₂₅N₃O₂ 106b 

339.43 g/mol C₂₀H₂₅N₃O₂

Compound 81b: N¹-(2-aminophenyl)-N⁷-(2-methoxyphenyl)heptanediamide

A solution of 80b (1.86 g, 7.00 mmol), EDC.HCl (1.33 g, 7.00 mmol), HOBt(1.071 g, 7.00 mmol) in DMF (10 mL) was added dropwise to a stirred andcooled solution of 1,2-phenylenediamine (1.50 g, 14.0 mmol) in DMF (10mL). After stifling the reaction for 1 hour at room temperature, thesolvent was removed and the residue was taken up in 150 mL ethylacetateand washed with two times with 20 mL 5% citric acid, two times with 20mL saturated sodium bicarbonate and two times with saturated NaCl. Afterdrying over MgSO₄ the solvent was evaporated and the crude product wasrecrystallized from water/ethanol.

The reaction yielded 1.366 g (3.85 mmol, 55%) of 81b as a yellow solid:TLC: R_(f)=0.53 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z (%)=356.0(100, [M+H]⁺, calc. 356.2), 378.0 (20, [M+Na]⁺, calc. 378.2); ¹H-NMR(500 MHz, D₄-MeOH): δ 1.46-1.53 (m, 2H), 1.71-1.80 (m, 4H), 2.41-2.47(m, 4H), 3.85 (s, 3H), 6.67-6.72 (m, 1H), 6.82-6.92 (m, 2H), 6.97-7.02(m, 2H), 7.03-7.10 (m, 2H), 7.90 (m, 1H).

Compound 82b: N¹-(4-aminophenyl)-N⁷-(2-methoxyphenyl)heptanediamide

A solution of 2-methoxyaniline (33 μL, 286 μmol) in DMF (5 mL) was addeddropwise to a cooled solution of P3 (100 mg, 286 μmol), EDC.HCl (55 mg,286 μmol), HOBt (44 mg, 286 μmol) in DMF (5 mL). After stifling for 3hours, the solvent was removed in vacuo, and the residue was trituratedwith 10 mL DCM/TFA (1:1) and stirred for 2 hours at room temperature.The solvent was removed and the residue was recrystallized fromacetonitrile/water.

The reaction yielded 55 mg (154 μmol, 54%) of 82b as a colorless solid:TLC: R_(f)=0.26 (DCM/MeOH 20:1); RP-HPLC: R_(t)=28 min; MS (MALDI-ToF,CHCA): m/z (%)=356.3 (100, [M+H]⁺, calc. 356.2), 378.2 (28, [M+Na]⁺,calc. 378.2); ¹H-NMR (500 MHz, D₆-DMSO): δ 1.30-1.35 (m, 2H), 158-1.64(m, 4H), 2.29-2.39 (m, 4H), 3.80 (s, 3H), 6.85-6.88 (m, 1H), 7.00-7.06(m, 2H), 7.14 (AA′BB′, 2H), 7.62 (AA′BB′, 2H), 7.90 (m, 1H), 9.00 (s,1H), 9.97 (s, 1H).

Compound 102b: N¹-(2-aminophenyl)-N⁷-o-tolylheptanediamide

A solution of 100b (200 mg, 0.79 mmol), EDC.HCl (152 mg, 0.79 mmol),HOBt (121 mg, 0.79 mmol) in DMF (5 mL) was added dropwise to a stirredand cooled solution of 1,2-phenylenediamine (430 mg, 4.00 mmol) in DMF(5 mL). After stifling the reaction for 1 hour at room temperature,water was added until a white solid precipitated, which was collected byfiltration and recrystallized from ethanol/water.

This reaction yielded 186 g (0.55 mmol, 69%) of 102b as a colorlesssolid: TLC: R_(f)=0.21 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z(%)=340.4 (95, [M+H]⁺, calc. 340.2), 362.4 (43, [M+Na]⁺, calc. 362.2);¹H-NMR (500 MHz, D₆-DMSO): δ 1.37-1.42 (m, 2H), 1.59-1.65 (m, 4H),2.32-2.36 (m, 4H), 3.32 (s, 3H), 4.80 (br. s, 2H), 6.49-6.55 (m, 1H),6.70-6.72 (m, 1H), 6.87-6.89 (m, 1H), 7.06-7.19 (m, 4H), 7.35-7.37 (m,1H), 9.08 (s, 1H), 9.22 (s, 1H).

Compound 106b: N¹-(2-aminophenyl)-N⁷-p-tolylheptanediamide

A solution of 101b (750 mg, 3.00 mmol), EDC.HCl (573 mg, 3.00 mmol),HOBt (460 mg, 3.00 mmol) in DMF (20 mL) was added dropwise to a stirredand cooled solution of 1,2-phenylenediamine (1.62 mg, 15.0 mmol) in DMF(10 mL). After stifling the reaction for 2 hours at room temperature,water was added until a white solid precipitated, which was collected byfiltration and recrystallized from ethanol/water.

This reaction yielded 742 mg (2.19 mmol, 73%) of 101b as a colorlesssolid: TLC: R_(f)=0.53 (DCM/MeOH 20:1); MS (MALDI-ToF, CHCA): m/z(%)=340.3 (100, [M+H]⁺, calc. 340.2), 362.4 (24, [M+Na]⁺, calc. 362.2);¹H-NMR (500 MHz, D₆-DMSO): δ 1.33-1.36 (m, 2H), 1.60-1.65 (m, 4H),2.25-2.33 (m, 4H), 3.35 (s, 3H), 4.80 (br. s, 2H), 6.50-6.54 (m, 1H),6.70-6.72 (m, 1H), 6.87-6.90 (m, 1H), 7.07-7.09 (AA′BB′, 2H), 7.13-7.16(m, 1H), 7.45-7.47 (AA′BB′, 2H), 9.05 (s, 1H), 9.76 (s, 1H).

Example 3—Histone Compositions of Active & Repressed Frataxin Alleles

To assess whether histone modifications play a role in gene silencing inFRDA, the histone acetylation state of the frataxin gene in an EpsteinBarr virus transformed lymphoid cell line derived from an FRDA patient(line GM15850, alleles with 650 and 1030 GAA.TTC repeats in the frataxingene, from the NIGMS Human Genetic Cell Repository, Coriell Institute,Camden, N.J.) was monitored by chromatin immunoprecipitation (ChIP) withantibodies to the acetylated forms of histones H3 and H4. Forcomparison, we used a similar cell line from a normal sibling of thispatient (line GM15851, normal range of repeats). As expected, the cellline from the FRDA patient has a markedly lower level (13±6%, range of20 determinations (Burnett et al. P.N.A.S. 103: 11497-502 (2006)) offrataxin mRNA compared to the cell line from the unaffected sibling, asdetermined by quantitative real time/reverse transcriptase PCR (qRT-PCR,see below). Primers that interrogate the chromatin regions upstream ordownstream of the GAA.TTC repeats in the first intron of the frataxingene, as well as the promoter element, were used in the ChIPexperiments, with the levels of immunoprecipitated DNA quantified byreal-time PCR (FIG. 1A). There was no difference in the expression ofglyceraldehyde-3-phosphodehydrogenase (GAPDH) mRNA between the two celllines, and GAPDH was used as a recovery standard in the ChIPexperiments. The coding region of active frataxin alleles in the GM15851cell line is enriched in histones acetylated at H3-K9, H3-K14, H4-K5,H4-K8, H4-K12, and H4-K16, compared to the inactive alleles in theGM15850 FRDA cell line, which are clearly depleted in these histonemodifications. No significant differences in the levels of histoneacetylation were found on the frataxin promoter in the two cell lines.Additionally, we examined the methylation status of H3-K9 withantibodies to mono-, di- and tri-methylated H3-K9, and H3-K9 is highlytrimethylated in the FRDA cell line compared to the normal cell line(FIG. 1B). Along with hypoacetylation, trimethylation of H3-K9 is ahallmark of heterochromatin (Elgin & Grewal, Curr. Biol. 13:R895-8(2003)). Thus, the histone postsynthetic modification states within thecoding region of inactive frataxin alleles are consistent with achromatin-mediated mechanism as the cause of gene silencing in FRDA(Saveliev et al. Nature 422:909-13 (2003)).

Example 4—Effect of Histone Deacetylase Inhibitors on Frataxin GeneExpression

To further assess the possibility that gene silencing at expandedGAA.TTC frataxin alleles is due to histone deacetylation andheterochromatin formation, the effects of a series of commercial HDACinhibitors on the levels of histone acetylation and frataxintranscription in the FRDA and normal lymphoid cell lines were monitoredusing antibodies to the acetylated forms of histones H3 and H4. Theinhibitors tested included the hydoxamic acids trichostatin A, suberoylbis-hydroxamic acid (SBHA), and suberoylanilide hydroxamic acid (SAHA);the benzamide-type SAHA derivative BML-210 (Wong et al. J. Am. Chem.Soc. 125:5586-7 (2003)); and the small caboxylate valproic acid. Thestructures of SAHA, PAOA (compound 4b), and SAOA (compound 4c, BML-210)are illustrated below:

Results in FIG. 2A indicate that all of the HDAC inhibitors increasedthe levels of total acetylated histones in the FRDA cell line when usedat their reported IC₅₀ value for HDAC inhibition. Cyclic peptideinhibitors were also tested, but were found to be highly cytotoxic tothe lymphoid cells, and thus not pursued. Each of the HDAC inhibitorswas also tested for effects on frataxin mRNA levels in the FRDA cellline by qRT-PCR (at the IC₅₀ values), and only BML-210 increased thelevel of frataxin mRNA ˜2-fold (FIG. 2B). The levels of GAPDH mRNA werenot changed by the HDAC inhibitors and were used for normalization inall qRT-PCR experiments. Over the concentration range necessary for HDACinhibition (1 to 5 μM), BML-210 is not cytotoxic to the lymphoid celllines (determined by trypan blue exclusion) and does not markedly affectcell growth rates. The structurally related compound SAHA had no effecton frataxin transcription and SBHA had a negative effect (50% decrease),even though both compounds were more effective HDAC inhibitors thanBML-210 (FIG. 2A).

Example 5—Evaluation of HDAC Inhibitors

To optimize the activity of BML-210, compound 4c(N¹-(2-aminophenyl)-N⁸-phenyloctanediamide, Wong et al. J. Am. Chem.Soc. 125:5586-7 (2003)) and a series of related analogues (see Table 4)were synthesized by a facile two-step protocol (see Example 2).Derivatives were designed to explore the length of the linker regionbetween the two ring systems (four, five and six methylenes), the natureof the rings (phenyl, pyridine, quinoline), and the type and position ofring substituents (methyl and methoxy groups, etc., Table 4).

TABLE 4 Relative Activities of Histone Deacetylase InhibitorsFold-change¹ Compound (IC₅₀)²  4a

1.4 ± 0.06 (238 μM)  4b

2.5 ± 0.24  (78 μM)  4c

1.4 ± 0.06  (87 μM)  5b

1.4 ± 0.15 (204 μM)  6b

1.5 ± 0.13 (500 μM)  6c

2.1 ± 0.15  (85 μM) Fold-Change Compound (IC₅₀)  7b

2.6 ± 0.14 123 μM)  7c

2.0 ± 0.08 (186 μM)  8b

2.6 ± 0.14 (140 μM)  8c

2.0 ± 0.08  (99 μM)  9b

2.3 ± 0.11  (54 μM)  9c

1.8 ± 0.07 (470 μM) 10b

2.5 ± 0.17 (438 μM) 11b

3.0 ± 0.17  (17 μM) 12b

2.5 ± 0.17  (84 μM) 13b

2.4 ± 0.10  (91 μM) 14b

1.8 ± 0.12  (>1 mM) 15b

1.5 ± 0.06 (387 μM) 16b

3.1 ± 0.19  (14 μM) ¹Fold-change of frataxin mRNA in affected GM15850cells, normalized to GAPDH mRNA, were determined in triplicate byreal-time quantitative RT-PCR after incubation with each compound at 5μM for 96 h. Values are relative to untreated control cells. ²IC₅₀values (in parenthesis below fold-change values) were determined bytotal histone deacetylation inhibition in a HeLa nuclear extract.

The compounds were tested for their effects on frataxin mRNA levels inthe FRDA cell line by qRT-PCR and for their activity as HDAC inhibitorsin a HeLa nuclear extract (Table 4). The IC₅₀ values, representing thegeneral HDAC inhibitory activities of these compounds, range from 14 μM(compound 16b) to >1000 μM (compound 14b, Table 4). The same IC₅₀ valueswere obtained with an extract from FRDA lymphoid cells for several ofthe compounds (not shown). For activation of transcription, each of thecompounds was tested at a concentration of 5 μM in culture medium for 96hours. Importantly, and in contrast to the common HDAC inhibitors suchas SAHA and TSA, none of the compounds affected the viability of thelymphoid cell lines (at concentrations required for transcriptionalactivation). Compounds with six (4c and derivatives) or four (4a)methylene groups in the linker region are less potent transcriptionalactivators than the corresponding pimeloylanilide derivatives (4b,N¹-(2-aminophenyl)-N⁷-phenylheptanediamide (Wong et al. J. Am. Chem.Soc. 125:5586-7 (2003)), and derivatives, Table 4), and amino- ormethoxy-substitutions at the ortho- and para-positions are mosteffective in increasing levels of frataxin mRNA. The quinolinederivatives of pimeloylanilide are also highly active (compounds 11b and12b). The symmetric diamino compound 16b, N¹,N⁷-bis(2-aminophenyl)heptanediamide, is the most effective compound in the FRDA cell line(3.1-fold increase in frataxin mRNA at 5 μM and 3.5-fold increase at 10μM). 16b exhibits an IC₅₀ value of 14 μM in a HeLa nuclear extract HDACinhibition assay, compared to an IC₅₀ of 87 μM for 4c and 78 μM for 4b.There is no apparent correlation between total HDAC inhibition activityand the ability of the compounds to activate transcription of thefrataxin gene in live cells. These findings are in accord with theobservation that common class I and II HDAC inhibitors have no effect onfrataxin transcription (FIG. 2B). Since the nuclear extracts containseveral HDAC enzymes, in many different multi-protein complexes, thestandard HDAC inhibition assay only provides an overall measure ofinhibitory activity for the sum of all these enzymatic activities. Weinterpret these results to indicate that the general HDAC assay does notreflect the IC₅₀ for the true target enzyme involved in silencing thefrataxin gene.

Example 6—HDAC Inhibitors Increase Frataxin Protein Levels

Since the primary transcripts of pathogenic frataxin alleles containlong GAA repeat RNA sequences, it is conceivable that these RNAs may notbe correctly processed and increases in frataxin protein may not beobserved on treatment with HDAC inhibitors. To test whether the HDACinhibitors lead to increased levels of frataxin protein in treatedlymphoid cells, total cellular proteins were subjected to SDS-PAGE andwestern blotting with anti-frataxin or anti-actin antibodies (FIG. 3). A˜3-fold increase in frataxin protein is observed with 4c (BML-210) at 5μM, and a similar increase in frataxin protein was observed with 4b at2.5 μM in the FRDA cells. These increases in frataxin protein equal orexceed the observed increases in frataxin mRNA in cultured cells (Table4).

Example 7—HDAC Inhibitors Increase Frataxin mRNA in Primary Lymphocytesfrom FRDA Patients

Frataxin protein deficiency in the human disease affectsnon-proliferating cell types (neuronal cells and cardiomyocytes). Whilethese human cells are not readily available for experimentation, primarylymphocytes can be obtained from donor blood, and lymphocytes that arenot treated with cytokines do not divide in culture under the conditionsof our experiments. We thus tested the effect of HDAC inhibitors onfrataxin mRNA levels in primary lymphocytes obtained from FRDA patientsand carrier or normal relatives of these patients (under an approvedHuman Subjects Protocol, with appropriate informed consent). Lymphocyteswere isolated by Ficoll gradient centrifugation, and cells wereincubated in culture for 16 h prior to the addition of 2.5 or 5 μM 4b or4c to the culture medium; cells were harvested and RNA purified after anadditional 48 hours in culture. Similar to the established cell lines,the HDAC inhibitors did not affect viability of primary lymphocytes overthis time period. Lymphocytes from affected individual S had 33±2% ofthe level of frataxin mRNA compared to lymphocytes from his/herhomozygous normal sibling A (FIG. 4A). Neither compound affected thelevels of GAPDH mRNA in cells from either individual, while incubationfor two days in culture with 4b and 4c markedly stimulated frataxin mRNAsynthesis in lymphocytes from the affected individual (FIG. 4A). Therelative levels of frataxin mRNA increased by 1.8-fold (80% increase)with 5 μM 4c, and by 2.3-fold (130% increase) with 5 μM 4b in FRDAlymphocytes. 4b had a smaller effect (38% increase) in lymphocytes fromthe unaffected sibling, while 4c had no positive effect on frataxin mRNAin these cells. Importantly, 4b increased the frataxin mRNA level inlymphocytes from the affected individual to ˜80% of that in lymphocytesfrom the unaffected individual.

We next compared the transcriptional activities of five of the mostactive HDAC inhibitors identified in the established cell line inprimary lymphocytes from a heterozygous carrier (subject C, frataxinmRNA normalized to 100%) and FRDA patient AC (FIG. 4B). Again, 4b washighly active in increasing frataxin mRNA levels in cells obtained fromcarrier C and affected AC, bringing the frataxin mRNA level in the FRDAlymphocytes to ˜160% of that found in the untreated carrier lymphocytes.Compounds 13b and 16b were also active and brought the frataxin mRNAlevel in the FRDA lymphocytes to that found in lymphocytes from theunaffected carrier. Unlike the established FRDA cell line (Table 4),compounds 7b and 8c were relatively inactive in primary lymphocytes.Also in contrast to results with the established cell line where 16b ismost active, 4b was found to be the most active compound in primarylymphocytes, and we thus pursued 4b in subsequent studies. We nexttested the effects of increasing concentrations of 4b on frataxin mRNAlevels in lymphocytes from two sibling FRDA patients J and M, andcarrier relative D (with frataxin mRNA levels normalized to 100% in thecarrier lymphocytes, FIG. 4C). 4b increased the levels of frataxin mRNAin each of the tested lymphocyte populations, and the level of frataxinmRNA in the FRDA patient lymphocytes was increased to at least that ofthe carrier. Notably, frataxin mRNA was nearly doubled in the carrier,suggesting that the inactive frataxin allele has been nearly completelyre-activated. While differences in the fold-increases in frataxin mRNAare observed with 4b in primary lymphocytes from different donors(compare FIGS. 4A-C), this compound consistently increases frataxin mRNAin FRDA and carrier lymphocytes obtained from 12 out of 12 families, andin each instance the frataxin mRNA level in the FRDA lymphocytes isincreased to approximately that of untreated lymphocytes from a carrierrelative. We have thus obtained a level of gene activation thatrepresents a therapeutically useful increase in frataxin mRNA. We notethat the HDAC inhibitors are more effective in primary lymphocytes thanin the FRDA cell line, and this difference may be related to the moresevere silencing of the frataxin gene observed in the FRDA cell line.

Example 8—HDAC Inhibitors Act Directly on the Frataxin Gene

To assess whether the HDAC inhibitors act directly on the histoneacetylation state of the frataxin gene, we performed ChIP experimentsafter treatment of FRDA cells with the HDAC inhibitor 4b (at 5 μM for 96h) and analyzed histone acetylation on the chromatin region immediatelyupstream of the GAA repeats. Insufficient yields of cells precludeperforming this experiment with lymphocytes from donor blood. Since theregion immediately upstream of the GAA repeats showed the most strikingdifference in histone acetylation between the two cell lines (FIG. 1A),ChIP assays were performed with this probe. Similar ˜2.5 to 3-foldincreases in frataxin transcription (Table 4) and acetylation at H3-K14,H4-K5 and H4-K12 are observed with 4b in these cells (FIG. 5A). Nosignificant changes in acetylation are observed at H3-K9, H4-K8, orH4-K16. To demonstrate the specificity of the effect of 4b on histoneacetylation at the frataxin gene, we performed similar ChIP experimentsafter treatment of FRDA cells with two common HDAC inhibitors (TSA andSAHA) that had no effect frataxin transcription (FIG. 2B). When theregion immediately upstream of the GAA repeats in the frataxin gene wasprobed after FRDA cells were incubated with these compounds for 96 h, nosignificant effects on histone acetylation were observed (FIG. 5B).These data suggest that 4b directly inhibits an as yet unidentified HDACenzyme(s) associated with the frataxin gene, thus resulting in increasedlevels of acetylated histones by the action of an associated histoneacetyltransferase, ultimately leading to increases in frataxintranscription.

Example 9—Additional Selected HDAC Inhibitors

The following additional HDAC inhibitors were synthesized and tested asdescribed above.

KJ-81b: N¹-(2-aminophenyl)-N⁷-(2-methoxyphenyl)heptanediamide

KJ-82b: N¹-(4-aminophenyl)-N⁷-(2-methoxyphenyl)heptanediamide

KJ-102b: N¹-(2-aminophenyl)-N⁷-o-tolylheptanediamide

KJ-106b: N¹-(2-aminophenyl)-N⁷-p-tolylheptanediamide

The structures of these compounds and IC₅₀ for histone deacetylaseinhibition are summarized in Table 5.

TABLE 5 IC₅₀ Values of Selected Inhibitors Salt TFA- (IC₅₀) StructureMol. Wt. Formula adduct KJ-81b  (37 μM)

355.43 g/mol C₂₀H₂₅N₃O₃ + KJ-82b  (54 μM)

355.43 g/mol C₂₀H₂₅N₃O₃ + KJ-102b (not determined)

338,43 g/mol C₂₀H₂₅N₃O₂ + KJ-106b (64 μM)

339.43 g/mol C₂₀H₂₅N₃O₂ +

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Other Embodiments

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby incorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. As used herein and inthe appended claims, the singular forms “a,” “an,” and “the” includeplural reference unless the context clearly dictates otherwise. Thus,for example, a reference to “an antibody” includes a plurality (forexample, a solution of antibodies or a series of antibody preparations)of such antibodies, and so forth. Under no circumstances may the patentbe interpreted to be limited to the specific examples or embodiments ormethods specifically disclosed herein. Under no circumstances may thepatent be interpreted to be limited by any statement made by anyExaminer or any other official or employee of the Patent and TrademarkOffice unless such statement is specifically and without qualificationor reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

While the invention has been described in conjunction with the detaileddescription, the foregoing description is intended to illustrate and notlimit the scope of the invention, which is defined by the scope of theappended claims. Other aspects, advantages, and modifications are withinthe scope of the following claims.

What is claimed is:
 1. A method of treating, or delaying the onset ofHuntington's disease in a mammal comprising administering to the mammala compound of formula I in an amount effective to alter the level ofhistone acetylation in the mammal and increase frataxin mRNA inlymphocytes from Friedreich's ataxia patients, wherein formula I is:

wherein: n is 2 to about 10; R¹ is aryl or heteroaryl; R² is aryl orheteroaryl; R^(a) and R^(b) are each independently H, alkyl, aryl,heteroaryl, or a nitrogen protecting group; wherein any alkyl, aryl orheteroaryl is optionally substituted with 1 to 3 substituents selectedfrom the group consisting of hydroxy, amino, nitro, cyano, halo, alkyl,trifluoromethyl, alkoxy, aryl, and NR^(c)R^(d); wherein R^(c) and R^(d)are each independently hydrogen, alkyl, or C(═O)OR^(e) wherein R^(e) isH or alkyl; or a salt thereof.
 2. The method of claim 1, furthercomprising identifying the mammal as one suffering from, or at risk for,Huntington's disease.
 3. The method of claim 1, wherein the mammal is ahuman.
 4. The method of claim 1, wherein the compound of formula I has astructure selected from the group consisting of: a salt of a compoundthereof; and any combination thereof.